Tgf-beta receptor type ii variants and uses thereof

ABSTRACT

In certain aspects, the present disclosure relates to polypeptides comprising a truncated, ligand-binding portion of the extracellular domain of TβRII polypeptide useful to selectively antagonize a TβRII ligand. The disclosure further provides compositions and methods for use in treating or preventing TGFβ associated disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/777,546, filed Jan. 30, 2020 (now allowed), which is a divisional ofU.S. application Ser. No. 16/393,277, filed Apr. 24, 2019 (now allowed),which is a continuation of Ser. No. 15/714,015, filed Sep. 25, 2017 (nowU.S. Pat. No. 10,316,076), which is a continuation of U.S. applicationSer. No. 15/044,883, filed on Feb. 16, 2016 (now U.S. Pat. No.9,809,637), which is a continuation of U.S. application Ser. No.14/465,182, filed on Aug. 21, 2014 (now abandoned), which claims thebenefit of priority to U.S. Provisional Application Ser. No. 61/906,849,filed Nov. 20, 2013, 61/906,270, filed Nov. 19, 2013, and 61/868,713,filed Aug. 22, 2013. The specifications of each of the foregoingapplications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 12, 2021, isnamed 1848179-0002-071-106_Seq.txt and is 138,915 bytes in size.

Background of the Invention

Members of the transforming growth factor-beta (TGFβ) superfamily arepleiotropic cytokines involved in essential cellular functions such asproliferation, differentiation, apoptosis, motility, extracellularmatrix production, tissue remodeling, angiogenesis, immune response,cell adhesion, and also play a key role in pathophysiology of diseasestates as different as chronic inflammatory conditions and cancer.Members of the TGFβ superfamily have been classified into major familygroupings, which include TGFβs, bone morphogenetic proteins (BMP),osteogenic proteins (OP), growth and differentiation factors (GDF),inhibins/activins, mullerian inhibitory substances (MIS) and glialderived neurotrophic factors (GDNF).

TGFβ superfamily members transduce their signals across the plasmamembrane by inducing the formation of heteromeric complexes of specifictype I and type II serine/threonine kinase receptors, which in turnactivate a particular subset of SMAD proteins (some inhibitory and someexcitatory). The SMAD molecule compounds relay the signals into thenucleus where they direct transcriptional responses in concert withother proteins.

Dysfunctional TGFβ superfamily signaling has been linked to severalclinical disorders including cancer, fibrosis, bone diseases, diabeticnephropathy, as well as chronic vascular diseases such asatherosclerosis.

Thus, it is an object of the present disclosure to provide compositionsand methods for modulating TGFβ superfamily signaling.

SUMMARY OF THE INVENTION

In part, the disclosure provides TβRII polypeptides and the use of suchTβRII polypeptides as selective antagonists for GDF15, TGFβ1 or TGFβ3.As described herein, polypeptides comprising part or all of the TβRIIextracellular domain (ECD), with or without additional mutations, bindto and/or inhibit GDF15, TGFβ1 or TGFβ3 with varying affinities. Thus,in certain aspects, the disclosure provides TβRII polypeptides for usein selectively inhibiting TGFβ superfamily associated disorders.

In certain aspects, the disclosure provides polypeptides comprisingmutations and/or truncations in the extracellular domain of TβRII. Incertain aspects, the disclosure provides a TβRII fusion polypeptidecomprising a first amino acid sequence from the extracellular domain ofTβRII and a heterologous amino acid sequence, wherein the first aminoacid sequence comprises or consists of an amino acid sequence at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% identical or identical to a) a sequencebeginning at any of positions 23 to 35 of SEQ ID NO: 5 and ending at anyof positions 153 to 159 of SEQ ID NO: 5 or b) a sequence beginning atany of positions 23 to 60 of SEQ ID NO: 6 and ending at any of positions178 to 184 of SEQ ID NO: 6.

In certain aspects the disclosure provides polypeptides comprising awild-type or altered and/or truncated extracellular domain of TβRIIfused to at least a portion of the Fc domain of a human IgG2. Thus incertain aspects, the disclosure provides a TβRII fusion polypeptidecomprising a first amino acid sequence from the extracellular domain ofTβRII and a heterologous amino acid sequence, wherein the first aminoacid sequence comprises or consists of an amino acid sequence at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% identical or identical to a) a sequencebeginning at any of positions 23 to 35 of SEQ ID NO: 5 and ending at anyof positions 153 to 159 of SEQ ID NO: 5 or b) a sequence beginning atany of positions 23 to 60 of SEQ ID NO: 6 and ending at any of positions178 to 184 of SEQ ID NO: 6, and wherein the polypeptide comprises asecond polypeptide sequence that comprises at least a constant domain ofa human IgG2 and may optionally comprise or consist of an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% identical to SEQID NO: 19, and wherein an linker is optionally positioned between thefirst polypeptide and the second polypeptide. An example of the isprovided as SEQ ID NO:50 and is encoded by the nucleic acid sequence ofSEQ ID NO:51. In certain embodiments, the disclosure providespolypeptides with an amino acid sequence that comprises or consists ofan amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%.98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:50.In certain embodiments, the disclosure provides polypeptides that areencoded by a nucleic acid sequence that comprises or consists of anucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%.98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:51.

In some embodiments, the first amino acid sequence comprises or consistsof the sequence beginning at position 23 of SEQ ID NO: 5 and ending atposition 159 of SEQ ID NO: 5. In some embodiments, the first amino acidsequence comprises or consists of the sequence beginning at position 29of SEQ ID NO: 5 and ending at position 159 of SEQ ID NO: 5. In someembodiments, the first amino acid sequence comprises or consists of thesequence beginning at position 35 of SEQ ID NO: 5 and ending at position159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequencecomprises or consists of the sequence beginning at position 23 of SEQ IDNO: 5 and ending at position 153 of SEQ ID NO: 5. In some embodiments,the first amino acid sequence comprises or consists of the sequencebeginning at position 29 of SEQ ID NO: 5 and ending at position 153 ofSEQ ID NO: 5. In some embodiments, the first amino acid sequencecomprises or consists of the sequence beginning at position 35 of SEQ IDNO: 5 and ending at position 153 of SEQ ID NO: 5.

In some embodiments, the first amino acid sequence comprises or consistsof the sequence beginning at position 23 of SEQ ID NO: 6 and ending atpositions 184 of SEQ ID NO: 6. In some embodiments, the first amino acidsequence comprises or consists of the sequence beginning at position 29of SEQ ID NO: 6 and ending at position 184 of SEQ ID NO: 6. In someembodiments, the first amino acid sequence comprises or consists of thesequence beginning at position 23 of SEQ ID NO: 6 and ending at position178 of SEQ ID NO: 6. In some embodiments, the first amino acid sequencecomprises or consists of the sequence beginning at position 29 of SEQ IDNO: 6 and ending at position 178 of SEQ ID NO: 6.

In some embodiments, the first amino acid sequence comprises or consistsof a sequence that has a D at the position corresponding to position 36of SEQ ID NO: 47 and/or a K at the position corresponding to position 76of SEQ ID NO: 47.

In certain aspects, the disclosure provides a TβRII fusion polypeptidecomprising a first amino acid sequence at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical or identical to the sequence of SEQ ID NO: 7 or SEQID NO: 13, or active fragment thereof, and a second heterologousportion, wherein the first amino acid sequence has a D at the positioncorresponding to position 36 of SEQ ID NO: 47 and/or a K at the positioncorresponding to position 76 of SEQ ID NO: 47.

In some embodiments, the first amino acid sequence comprises anN-terminal truncation of 1-12 amino acids corresponding to amino acids1-12 of SEQ ID NO: 7 or 1-37 amino acids corresponding to amino acids1-37 of SEQ ID NO: 13. In some embodiments, the first amino acidsequence comprises an N-terminal truncation of 6 amino acidscorresponding to amino acids 1-6 of SEQ ID NO: 7 or SEQ ID NO: 13. Insome embodiments, the first amino acid sequence comprises an N-terminaltruncation of 12 amino acids corresponding to amino acids 1-12 of SEQ IDNO: 7 or 37 amino acids corresponding to amino acids 1-37 of SEQ ID NO:13. In some embodiments, the first amino acid sequence comprises aC-terminal truncation of 1-6 amino acids corresponding to amino acids137-132 of SEQ ID NO: 7 or amino acids 162-157 of SEQ ID NO: 13. In someembodiments, the first amino acid sequence comprises a C-terminaltruncation of 6 amino acids corresponding to amino acids 132-137 of SEQID NO: 7 or amino acids 157-162 of SEQ ID NO: 13. In some embodiments,the first amino acid sequence comprises an insertion corresponding toSEQ ID NO: 18 between the residues corresponding to positions 117 and118 of SEQ ID NO: 47.

In some embodiments, the heterologous portion comprises one or morepolypeptide portions that enhance one or more of: in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Insome embodiments, the heterologous portion comprises a polypeptideportion selected from: an immunoglobulin Fc domain and a serum albumin.In a further embodiment, the immunoglobulin Fc domain is joined to theTβRII polypeptide by a linker.

In some embodiments, the polypeptide includes one or more modified aminoacid residues selected from: a glycosylated amino acid, a PEGylatedamino acid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety, andan amino acid conjugated to an organic derivatizing agent. In someembodiments, the polypeptide is glycosylated.

In certain aspects, the disclosure provides a TβRII fusion polypeptidecomprising a first amino acid sequence consisting of a portion of theextracellular domain of TβRII that comprises an amino acid sequence thatis at least 80%, at least 85%, at least 90%, or at least 95% identicalto an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, anda second heterologous portion. In certain aspects, the disclosureprovides a TβRII fusion polypeptide comprising a first amino acidsequence consisting of a portion of the extracellular domain of TβRIIthat comprises an amino acid sequence that is at least 96% identical toan amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and asecond heterologous portion. In certain aspects, the disclosure providesa TβRII fusion polypeptide comprising a first amino acid sequenceconsisting of a portion of the extracellular domain of TβRII thatcomprises an amino acid sequence that is at least 97% identical to anamino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and asecond heterologous portion. In certain aspects, the disclosure providesa TβRII fusion polypeptide comprising a first amino acid sequenceconsisting of a portion of the extracellular domain of TβRII thatcomprises an amino acid sequence that is at least 98% identical to anamino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and asecond heterologous portion. In certain aspects, the disclosure providesa TβRII fusion polypeptide comprising a first amino acid sequenceconsisting of a portion of the extracellular domain of TβRII thatcomprises an amino acid sequence that is at least 99% identical to anamino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and asecond heterologous portion. In certain aspects, the disclosure providesa TβRII fusion polypeptide comprising a first amino acid sequenceconsisting of a portion of the extracellular domain of TβRII thatcomprises an amino acid sequence is an amino acid sequence selected fromSEQ ID NOs: 7-17 and 47-49 and a second heterologous portion.

In certain aspects, the disclosure provides a polypeptide comprising orconsisting of an amino acid sequence that is at least 80%, at least 85%,at least 90%, or at least 95% identical to an amino acid sequenceselected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 orthe portion thereof with the leader sequence removed, e.g., apolypeptide comprising or consisting of an amino acid sequence that isat least 80%, at least 85%, at least 90%, or at least 95% identical toan amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58,59, 60, 61, and 62. In certain aspects, the disclosure provides apolypeptide comprising or consisting of an amino acid sequence that isat least 96% identical to an amino acid sequence selected from SEQ IDNOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereofwith the leader sequence removed, e.g., a polypeptide comprising orconsisting of an amino acid sequence that is at least 96% identical toan amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58,59, 60, 61, and 62. In certain aspects, the disclosure provides apolypeptide comprising or consisting of an amino acid sequence that isat least 97% identical to an amino acid sequence selected from SEQ IDNOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereofwith the leader sequence removed, e.g., a polypeptide comprising orconsisting of an amino acid sequence that is at least 97% identical toan amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58,59, 60, 61, and 62. In certain aspects, the disclosure provides apolypeptide comprising or consisting of an amino acid sequence that isat least 98% identical to an amino acid sequence selected from SEQ IDNOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereofwith the leader sequence removed, e.g., a polypeptide comprising orconsisting of an amino acid sequence that is at least 98% identical toan amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58,59, 60, 61, and 62. In certain aspects, the disclosure provides apolypeptide comprising or consisting of an amino acid sequence that isat least 99% identical to an amino acid sequence selected from SEQ IDNOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereofwith the leader sequence removed, e.g., a polypeptide comprising orconsisting of an amino acid sequence that is at least 99% identical toan amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58,59, 60, 61, and 62. In certain aspects, the disclosure provides apolypeptide comprising or consisting of an amino acid sequence selectedfrom SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or theportion thereof with the leader sequence removed, e.g., a polypeptidecomprising or consisting of an amino acid sequence selected from SEQ IDNOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.

In certain aspects, the disclosure provides a TβRII polypeptidecomprising of an amino acid sequence encoded by a nucleic acid thathybridizes under stringent conditions to a complement of a nucleotidesequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42and 44.

In each of the foregoing, the TβRII polypeptide may be selected that itdoes not include a full-length TβRII ECD. A TβRII polypeptide may beused as a monomeric protein or in a dimerized form. A TβRII polypeptidemay also be fused to a second polypeptide portion to provide improvedproperties, such as increased half-life or greater ease of production orpurification. A fusion may be direct or a linker may be inserted betweenthe TβRII polypeptide and any other portion. A linker may be structuredor unstructured and may consist of 1, 2, 3, 4, 5, 10, 15, 20, 30, 50 ormore amino acids, optionally relatively free of secondary structure. Insome embodiments, a TβRII polypeptide of the disclosure binds humanGDF15 with an equilibrium dissociation constant (K_(D)) less than 1×10⁻⁸M.

In some embodiments, a TβRII polypeptide of the disclosure has aglycosylation pattern characteristic of expression of the polypeptide inCHO cells.

In some embodiments, the disclosure provides a homodimer comprising twoTβRII polypeptides of the disclosure.

In some embodiments, the disclosure provides an isolated polynucleotidecomprising a coding sequence for the TβRII polypeptides of thedisclosure. In some embodiments, the disclosure provides a recombinantpolynucleotide comprising a promoter sequence operably linked to theisolated polynucleotide. In some embodiments, the disclosure provides acell transformed with an isolated polynucleotide or a recombinantpolynucleotide of the disclosure. In some embodiments, the cell is amammalian cell. In some embodiments, the cell is a CHO cell or a humancell. In some embodiments, the cell is an HEK-293 cell.

In certain aspects, the disclosure provides a pharmaceutical preparationcomprising the TβRII polypeptides or homodimers of the disclosure and apharmaceutically acceptable excipient.

In certain aspects, the disclosure provides a method of modulating theresponse of a cell to a TGFβ superfamily member, the method comprisingexposing the cell to a TβRII polypeptide or homodimer of the disclosure.

In certain aspects, the disclosure provides a method of treating adisease or condition associated with a TGFβ superfamily member in apatient in need thereof, the method comprising administering to thepatient an effective amount of the TβRII polypeptides or homodimers ofthe disclosure. In some embodiments, the TGFβ superfamily member isTGFβ1, TGFβ3 or GDF15. In some embodiments, the disease or condition isa cancer. In some embodiments, the cancer is selected from stomachcancer, intestinal cancer, skin cancer, breast cancer, melanoma, bonecancer and thyroid cancer.

In some embodiments, the disease or condition is a fibrotic or scleroticdisease or disorder. In some embodiments, the fibrotic or scleroticdisease or disorder is selected from scleroderma, atherosclerosis, liverfibrosis, diffuse systemic sclerosis, glomerulonephritis, neuralscarring, dermal scarring, radiation-induced fibrosis, hepatic fibrosis,and myelofibrosis.

In some embodiments, the disease or condition is heart disease.

In some embodiments, the disease or condition is selected fromhereditary hemorrhagic telangiectasia (HHT), Marfan syndrome,Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome,arterial tortuosity syndrome, pre-eclampsia, atherosclerosis,restenosis, and hypertrophic cardiomyopathy/congestive heart failure.

In certain aspects, the disclosure provides an antibody, or antigenbinding fragment thereof, that binds to GDF15 and blocks the interactionbetween GDF15 and TβRII. In certain aspects, the disclosure provides aGDF15 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 ora fragment thereof that binds TβRII, wherein the GDF15 polypeptide is atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%pure, with respect to protein contaminants.

In certain aspects, the disclosure provides a GDF15 polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereofthat binds to a TβRII polypeptide of the disclosure, wherein the GDF15polypeptide is at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% pure, with respect to protein contaminants.

In some embodiments, the GDF15 polypeptide binds TβRII with anequilibrium dissociation constant (K_(D)) of no greater than 10⁻⁸ M. Insome embodiments, the GDF15 polypeptide binds to a TβRII polypeptide ofthe disclosure with an equilibrium dissociation constant (K_(D)) of nogreater than 10⁻⁸ M.

In some embodiments, the GDF15 polypeptide is produced by expression inCHO cells.

In certain aspects, the disclosure provides a method of concentrating orpurifying GDF15, comprising contacting a sample containing GDF15 with aTβRII polypeptide of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of native precursor for human GDF15(NCBI reference seq: NP_004855.2). Solid underline indicates matureGDF15 (residues 197-308), with N-terminus determined by sequencing.Dotted underline denotes leader (residues 1-29).

FIG. 2 shows a nucleotide sequence encoding native precursor for humanGDF15. Solid underline indicates the sequence encoding mature GDF15(nucleotides 589-924), and dotted underline denotes the sequenceencoding the leader (nucleotides 1-87). A silent mutation (G456A) usedto disrupt a SfoI site in NM_004864.2 is double underlined.

FIG. 3 shows the amino acid sequence of native precursor for murineGDF15 (NP_035949.2). Solid underline indicates mature GDF15 (residues192-303), with N-terminus determined by sequencing. Dotted underlinedenotes leader (residues 1-30).

FIG. 4 shows a nucleotide sequence encoding native precursor for murineGDF15 (derived from NM_011819.2). Solid underline indicates the sequenceencoding mature GDF15 (nucleotides 574-909), and dotted underlinedenotes the sequence encoding the leader (nucleotides 1-90).

FIG. 5 shows the amino acid sequence of native precursor for the B(short) isoform of human TGFβ receptor type II (hTβRII) (NP_003233.4).Solid underline indicates the mature extracellular domain (ECD)(residues 23-159), and double underline indicates valine that isreplaced in the A (long) isoform. Dotted underline denotes leader(residues 1-22).

FIG. 6 shows the amino acid sequence of native precursor for the A(long) isoform of human TβRII (NP_001020018.1). Solid underlineindicates the mature ECD (residues 23-184), and double underlineindicates the splice-generated isoleucine substitution. Dotted underlinedenotes leader (residues 1-22).

FIG. 7 shows N-terminal alignment of hTβRII_(short) truncations (SEQ IDNOs: 64-67, respectively, in order of appearance) and theirhTβRII_(long) counterparts (SEQ ID NOs: 68-69 and 45-46, respectively,in order of appearance). The 25-amino-acid insertion present inhTβRII_(long) truncations is underlined. Note that the splicing processcauses the valine flanking the insertion site in the short isoform to bereplaced by an isoleucine in the long isoform. Boxed sequence denotesleader.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

Proteins described herein are the human forms, unless otherwisespecified. NCBI references for the proteins are as follows: human TβRIIisoform A (hTβRII_(long)), NP_001020018.1; human TβRII isoform B(hTβRII_(short)), NP_003233.4; human GDF15, NP_004855.2; murine GDF15,NP_035949.2. Sequences of native TβRII and GDF15 proteins from human andmouse are set forth in FIGS. 1-6.

The TGFβ superfamily contains a variety of growth factors that sharecommon sequence elements and structural motifs. These proteins are knownto exert biological effects on a large variety of cell types in bothvertebrates and invertebrates. Members of the superfamily performimportant functions during embryonic development in pattern formationand tissue specification and can influence a variety of differentiationprocesses, including adipogenesis, myogenesis, chondrogenesis,cardiogenesis, hematopoiesis, neurogenesis, and epithelial celldifferentiation. By manipulating the activity of a member of the TGFβfamily, it is often possible to cause significant physiological changesin an organism. For example, the Piedmontese and Belgian Blue cattlebreeds carry a loss-of-function mutation in the GDF8 (also calledmyostatin) gene that causes a marked increase in muscle mass. Grobet etal., Nat Genet. 1997, 17(1):71-4. Similarly, in humans, inactive allelesof GDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

TGFβ signals are mediated by heteromeric complexes of type I (e.g. TβRI)and type II (e.g. TβRII) serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins upon ligandstimulation (Massagué, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). Thesetype I and type II receptors are transmembrane proteins, composed of aligand-binding extracellular domain with cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine specificity. Type I receptors are essential forsignaling; and type II receptors are required for binding ligands andfor expression of type I receptors. Type I and II receptors form astable complex after ligand binding, resulting in phosphorylation oftype I receptors by type II receptors. TGFβ has three mammalianisoforms, TGFβ1, TGFβ2 and TGFβ, each with distinct functions in vivo.The binding of TGFβs to TβRII is a crucial step in initiating activationof the TGFβ signaling pathway, leading to phosphorylation of SMAD2, andtranslocation of the activated SMAD2/SMAD4 complex to the nucleus tomodulate gene expression.

Growth differentiation factor 15 (GDF15) is a member of the TGFβ family.Like other ligands in the TGFβ superfamily, which contain acharacteristic cysteine knot motif, mature GDF15 is synthesized with alarger prodomain (Harrison et al., Growth Factors 29:174, 2011; Shi etal., Nature 474:343, 2011) that is removed through cleavage by afurin-like protease at the canonical RXXR site to generate maturedimeric GDF15. GDF15 has been described in the literature as macrophageinhibitory cytokine-1 (MIC-1), placental bone morphogenic protein(PLAB), placental transforming growth factor beta (PTGFβ), prostatederived factor (PDF), and non-steroidal anti-inflammatory activatedgene-1 (NAG-1) reflecting the different functions that have been impliedfor this protein. GDF15 has been linked to several physiologic andpathologic conditions. For example, GDF15 is highly expressed in theplacenta, and is necessary for the maintenance of pregnancy. GDF15concentration is also notably increased in the serum of patients withprostate, colorectal, or pancreatic cancer, as well as glioma. GDF15 hasnot been shown biochemically to bind or interact directly with anyreceptor. The present disclosure relates in part to the discovery thatthe TGFβ type II receptor, TβRII, binds to GDF15 with high affinity andis a functional receptor for GDF15. TβRII fusion polypeptides, and otherpolypeptides containing a ligand-binding portion of TβRII aredemonstrated herein to inhibit GDF15-induced gene activation. The potentinhibition of GDF15 signaling provides evidence that TβRII is afunctional type II receptor for GDF15, opening a new avenue fortherapeutic interventions in this signaling pathway. Therefore, in part,the disclosure identifies a physiological, high-affinity receptor forGDF15 polypeptides.

Surprisingly, soluble TβRII polypeptides are shown herein to have highlyspecific, high-affinity binding for GDF15. TβRII is the known type IIreceptor for TGFβ and binds with high affinity to TGFβ1 and TGFβ3. HumanTβRII occurs naturally in at least two isoforms—A (long) and B(short)—generated by alternative splicing in the extracellular domain(ECD) (FIGS. 6 and 5 and SEQ ID NOS: 6 and 5). The long isoform has a25-amino-acid insertion and the splicing process causes the valineflanking the insertion site in the short isoform to be replaced by anisoleucine in the long isoform. Soluble receptor ectodomains canfunction as scavengers or ligand traps to inhibit ligand-receptorinteractions. Ligand traps such as soluble TβRII-Fc fusion proteinsincorporating the native TβRII extracellular domain (ectodomain) willfunction as pan-inhibitors against TβRII ligands, including, TGFβ 1,TGFβ3 and based on the findings disclosed herein, GDF15. While in sometherapeutic settings this broader spectrum of ligand-binding and signalinhibition may be advantageous, in other settings a more selectivemolecule may be superior. It is highly desirable for ligand traps suchas TβRII ectodomain polypeptides to exhibit selective ligand-bindingprofiles. The present disclosure relates to the surprising discoverythat polypeptides comprising a truncated portion of the extracellulardomain of TβRII and/or mutations within the extracellular domain havedifferential inhibitory effects on cell signaling by GDF15, TGFβ1 orTGFβ3. In part, the disclosure provides ligand traps, generated by aseries of mutations and/or truncations in the extracellular domain ofTβRII, that exhibit varying ligand-binding profiles distinct from thatof the native TβRII extracellular domain. The variant TβRII polypeptidesdisclosed herein provide advantageous properties relative to the nativefull-length extracellular domain, and may be used to selectively inhibitpathways mediated by the different TβRII ligands in vivo.

Thus, in certain aspects, the disclosure provides TβRII polypeptides asantagonists of GDF15, TGFβ1 or TGFβ3 for use in treating various GDF15-,TGFβ1- or TGFβ3-associated disorders. While not wishing to be bound toany particular mechanism of action, it is expected that suchpolypeptides act by binding to GDF15, TGFβ1 or TGFβ3 and inhibiting theability of these ligands to form ternary signaling complexes.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

2. TβRII Polypeptides

Naturally occurring TβRII proteins are transmembrane proteins, with aportion of the protein positioned outside the cell (the extracellularportion) and a portion of the protein positioned inside the cell (theintracellular portion). Aspects of the present disclosure encompassvariant TβRII polypeptides comprising mutations within the extracellulardomain and/or truncated portions of the extracellular domain of TβRII.As described above, human TβRII occurs naturally in at least twoisoforms—A (long) and B (short)—generated by alternative splicing in theextracellular domain (ECD) (FIGS. 6 and 5 and SEQ ID NOS: 6 and 5). SEQID NO: 7, which corresponds to residues 23-159 of SEQ ID NO: 5, depictsthe native full-length extracellular domain of the short isoform ofTβRII. SEQ ID NO: 13, which corresponds to residues 23-184 of SEQ ID NO:6, depicts the native full-length extracellular domain of the longisoform of TβRII. Unless noted otherwise, amino acid position numberingwith regard to variants based on the TβRII short and long isoformsrefers to the corresponding position in the native precursors, SEQ IDNO: 5 and SEQ ID NO:6, respectively.

In certain embodiments, the disclosure provides variant TβRIIpolypeptides. A TβRII polypeptide of the disclosure may bind to andinhibit the function of a TGFβ superfamily member, such as but notlimited to, GDF15, TGFβ1 or TGFβ3. TβRII polypeptides may include apolypeptide consisting of, or comprising, an amino acid sequence atleast 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to a truncated ECD domain of a naturallyoccurring TβRII polypeptide, whose C-terminus occurs at any of aminoacids 153-159 of SEQ ID NO: 5. TβRII polypeptides may include apolypeptide consisting of, or comprising, an amino acid sequence atleast 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to a truncated ECD domain of a naturallyoccurring TβRII polypeptide, whose C-terminus occurs at any of aminoacids 178-184 of SEQ ID NO: 6. Optionally, a TβRII polypeptide does notinclude more than 5 consecutive amino acids, or more than 10, 20, 30,40, 50, 52, 60, 70, 80, 90, 100, 150 or 200 or more consecutive aminoacids from a sequence consisting of amino acids 160-567 of SEQ ID NO: 5or from a sequence consisting of amino acids 185-592 of SEQ ID NO: 6.The unprocessed TβRII polypeptide may either include or exclude anysignal sequence, as well as any sequence N-terminal to the signalsequence. As elaborated herein, the N-terminus of the mature (processed)TβRII polypeptide may occur at any of amino acids 23-35 of SEQ ID NO: 5or 23-60 of SEQ ID NO: 6. Examples of mature TβRII polypeptides include,but are not limited to, amino acids 23-159 of SEQ ID NO: 5 (set forth inSEQ ID NO: 7), amino acids 29-159 of SEQ ID NO: 5 (set forth in SEQ IDNO: 9), amino acids 35-159 of SEQ ID NO: 5 (set forth in SEQ ID NO: 10),amino acids 23-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 11), aminoacids 29-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 48), amino acids35-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 47), amino acids 23-184of SEQ ID NO: 6 (set forth in SEQ ID NO: 13), amino acids 29-184 of SEQID NO: 6 (set forth in SEQ ID NO: 15), amino acids 60-184 of SEQ ID NO:6(set forth in SEQ ID NO: 10), amino acids 23-178 of SEQ ID NO: 6 (setforth in SEQ ID NO: 16), amino acids 29-178 of SEQ ID NO: 6 (set forthin SEQ ID NO: 49), and amino acids 60-178 of SEQ ID NO: 6 (set forth inSEQ ID NO: 47). Likewise, a TβRII polypeptide may comprise a polypeptidethat is encoded by nucleotides 73-465 of SEQ ID NO: 30, nucleotides73-447 of SEQ ID NO: 34, nucleotides 73-465 of SEQ ID NO: 38,nucleotides 91-465 of SEQ ID NO: 38, or nucleotides 109-465 of SEQ IDNO: 38, or silent variants thereof or nucleic acids that hybridize tothe complement thereof under stringent hybridization conditions(generally, such conditions are known in the art but may, for example,involve hybridization in 50% v/v formamide, 5×SSC, 2% w/v blockingagent, 0.1% N-lauroylsarcosine, and 0.3% SDS at 65° C. overnight andwashing in, for example, 5×SSC at about 65° C.). It will be understoodby one of skill in the art that corresponding variants based on the longisoform of TβRII will include nucleotide sequences encoding the 25-aminoacid insertion along with a conservative Val-Ile substitution at theflanking position C-terminal to the insertion. The TβRII polypeptidesaccordingly may include isolated extracellular portions of TβRIIpolypeptides, including both the short and the long isoforms, variantsthereof (including variants that comprise, for example, no more than 2,3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acid substitutions in thesequence corresponding to amino acids 23-159 of SEQ ID NO: 5 or aminoacids 23-184 of SEQ ID NO: 6), fragments thereof, and fusion proteinscomprising any of the foregoing, but in each case preferably any of theforegoing TβRII polypeptides will retain substantial affinity for atleast one of GDF15, TGFβ1 or TGFβ. Generally, a TβRII polypeptide willbe designed to be soluble in aqueous solutions at biologically relevanttemperatures, pH levels, and osmolarity.

In some embodiments, the variant TβRII polypeptides of the disclosurecomprise one or more mutations in the extracellular domain that conferan altered ligand binding profile. A TβRII polypeptide may include one,two, five or more alterations in the amino acid sequence relative to thecorresponding portion of a naturally occurring TβRII polypeptide. Insome embodiments, the mutation results in a substitution, insertion, ordeletion at the position corresponding to position 70 of SEQ ID NO: 5.In some embodiments, the mutation results in a substitution, insertion,or deletion at the position corresponding to position 110 of SEQ ID NO:5. Examples include, but are not limited to, an N to D substitution or aD to K substitution in the positions corresponding to positions 70 and110, respectively, of SEQ ID NO: 5. Examples of such variant TβRIIpolypeptides include, but are not limited to, the sequences set forth inSEQ ID NO: 8, SEQ ID NO:14, SEQ ID NO: 12 and SEQ ID NO: 17. A TβRIIpolypeptide may comprise a polypeptide or portion thereof that isencoded by nucleotides 73-483 of SEQ ID NO: 26, nucleotides 73-465 ofSEQ ID NO: 42 or silent variants thereof or nucleic acids that hybridizeto the complement thereof under stringent hybridization conditions.

In some embodiments, the variant TβRII polypeptides of the disclosurefurther comprise an insertion of 36 amino acids (SEQ ID NO: 18) betweenthe pair of glutamate residues (positions 151 and 152 of SEQ ID NO: 5,or positions 176 and 177 of SEQ ID NO: 6) located near the C-terminus ofthe human TβRII ECD, as occurs naturally in the human TβRII isoform C(Konrad et al., BMC Genomics 8:318, 2007).

The disclosure further demonstrates that TβRII polypeptides can bemodified to selectively antagonize TβRII ligands. Data presented hereshow that Fc fusion proteins comprising shorter N-terminally andC-terminally truncated variants of TβRII polypeptides displaydifferential inhibitory effects on cellular signaling mediated by GDF15,TGFβ1 and TGFβ3. Specifically, N-terminally truncated variants beginningat amino acids 29 or 35 of SEQ ID NO: 5 and carrying, respectively, a 6-or 12-amino acid N-terminal truncation of the extracellular domain, werefound to inhibit GDF15 most potently, TGFβ3 least potently and TGFβ1 toan intermediate degree, compared to the full length extracellular domainof the short isoform of TβRII. C-terminally truncated variants, endingat amino acid 153 of SEQ ID NO: 5 and carrying a 6-amino acid C-terminaltruncation of the extracellular domain had no substantial effect onligand binding and may therefore be used interchangeably with fulllength versions. An N to D substitution at the position corresponding toposition 70 of SEQ ID NO: 5, was found to inhibit TGFβ3 potently, haveintermediate effect on GDF15 and negligible effect on TGFβ1. The N70residue represents a potential glycosylation site. Further, an Fc fusionprotein comprising a D to K substitution at the position correspondingto position 110 of SEQ ID NO: 5, was found to inhibit GDF15 mostpotently, TGFβ1 least potently and TGFβ3 to an intermediate degreecompared to compared to the full length extracellular domain of theshort isoform of TβRII. The region around position 110 has not beenassociated with selectivity for the known TβRII ligands TGFβ 1, TGFβ2and TGFβ3. Thus, unexpectedly, TβRII polypeptides that contain mutationsin the ECD, such as but not limited to, N70D and D110K (the numbering ofthe residues corresponds to that of SEQ ID NO: 5) and/or begin betweenamino acids 29 and 35 and/or terminate between amino acid 153 and aminoacid 159 are all expected to be active and exhibit widely differentinhibitory potencies towards the different ligands. Any of thesetruncated variant forms may be desirable to use, depending on theclinical or experimental setting.

In certain embodiments, a TβRII polypeptide binds to GDF15, and theTβRII polypeptide does not show substantial binding to TGFβ1 or TGFβ3.In certain embodiments, a TβRII polypeptide binds to TGFβ1, and theTβRII polypeptide does not show substantial binding to GDF15 or TGFβ3.In certain embodiments, a TβRII polypeptide binds to TGFβ3, and theTβRII polypeptide does not show substantial binding to GDF15 or TGFβ1.Binding may be assessed using purified proteins in solution or in asurface plasmon resonance system, such as a Biacore™ system.

In certain embodiments, a TβRII polypeptide inhibits GDF15 cellularsignaling, and the TβRII polypeptide has an intermediate or limitedinhibitory effect on TGFβ1 or TGFβ3. In certain embodiments, a TβRIIpolypeptide inhibits TGFβ1 cellular signaling, and the TβRII polypeptidehas an intermediate or limited inhibitory effect on GDF15 or TGFβ3. Incertain embodiments, a TβRII polypeptide inhibits TGFβ3 cellularsignaling, and the TβRII polypeptide has an intermediate or limitedinhibitory effect on GDF15 or TGFβ 1. Inhibitory effect on cellsignaling can be assayed by methods known in the art.

Taken together, an active portion of a TβRII polypeptide may compriseamino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158of SEQ ID NO: 5, as well as variants of these sequences starting at anyof amino acids 24-35 of SEQ ID NO: 5. Similarly, an active portion of aTβRII polypeptide may comprise amino acid sequences 23-178, 23-179,23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 6, as well as variantsof these sequences starting at any of amino acids 24-60 of SEQ ID NO: 6.Exemplary TβRII polypeptides comprise amino acid sequences 29-159,35-159, 23-153, 29-153 and 35-153 of SEQ ID NO: 5 or amino acidsequences 29-184, 60-184, 23-178, 29-178 and 60-178 of SEQ ID NO: 6.Variants within these ranges are also contemplated, particularly thosehaving at least 80%, 85%, 90%, 95%, or 99% identity to the correspondingportion of SEQ ID NO: 5 or SEQ ID NO: 6. A TβRII polypeptide may beselected that does not include the sequence consisting of amino acids160-567 of SEQ ID NO:5 or amino acids 185-592 of SEQ ID NO:6.

As described above, the disclosure provides TβRII polypeptides sharing aspecified degree of sequence identity or similarity to a naturallyoccurring TβRII polypeptide. To determine the percent identity of twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The amino acid residues at corresponding amino acid positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue as the corresponding position in the second sequence,then the molecules are identical at that position (as used herein aminoacid “identity” is equivalent to amino acid “homology”). The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

In one embodiment, the percent identity between two amino acid sequencesis determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package. In a specific embodiment, the followingparameters are used in the GAP program: either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)). Exemplary parameters include usinga NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified,percent identity between two amino acid sequences is to be determinedusing the GAP program using a Blosum 62 matrix, a GAP weight of 10 and alength weight of 3, and if such algorithm cannot compute the desiredpercent identity, a suitable alternative disclosed herein should beselected.

In another embodiment, the percent identity between two amino acidsequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment betweentwo amino acid sequences can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.,6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is presented in terms of percent identity. In oneembodiment, amino acid sequence identity is performed using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

TβRII polypeptides may additionally include any of various leadersequences at the N-terminus. Such a sequence would allow the peptides tobe expressed and targeted to the secretion pathway in a eukaryoticsystem. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992).Alternatively, a native TβRII signal sequence may be used to effectextrusion from the cell. Possible leader sequences include nativeleaders, tissue plasminogen activator (TPA) and honeybee mellitin (SEQID NOs. 22-24, respectively). Examples of TβRII-Fc fusion proteinsincorporating a TPA leader sequence include SEQ ID NOs: 25, 27, 29, 31,33, 35, 37, 39, 41, and 43. Processing of signal peptides may varydepending on the leader sequence chosen, the cell type used and cultureconditions, among other variables, and therefore actual N-terminal startsites for mature TβRII polypeptides may shift by 1, 2, 3, 4 or 5 aminoacids in either the N-terminal or C-terminal direction. Examples ofTβRII-Fc fusion proteins include SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, as shown hereinwith the TβRII polypeptide portion underlined (see Examples). It will beunderstood by one of skill in the art that corresponding variants basedon the long isoform of TβRII will include the 25-amino acid insertionalong with a conservative Val-Ile substitution at the flanking positionC-terminal to the insertion.

In certain embodiments, the present disclosure contemplates specificmutations of the TβRII polypeptides so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagine-X-serine) (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the wild-type TβRIIpolypeptide (for O-linked glycosylation sites). A variety of amino acidsubstitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino aciddeletion at the second position) results in non-glycosylation at themodified tripeptide sequence. Another means of increasing the number ofcarbohydrate moieties on a TβRII polypeptide is by chemical or enzymaticcoupling of glycosides to the TβRII polypeptide. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on a TβRII polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the TβRII polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on TβRII polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of a TβRII polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, TβRII polypeptides for use inhumans will be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines, yeast cell lines with engineeredglycosylation enzymes, and insect cells are expected to be useful aswell.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of a TβRII polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, TβRII polypeptide variants which can act as either agonistsor antagonist, or alternatively, which possess novel activities alltogether. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, a TβRIIpolypeptide variant may be screened for ability to bind to a TβRIIligand, to prevent binding of a TβRII ligand to a TβRII polypeptide orto interfere with signaling caused by a TβRII ligand. The activity of aTβRII polypeptide or its variants may also be tested in a cell-based orin vivo assay, particularly any of the assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a TβRII polypeptidecomprising an extracellular domain of a naturally occurring TβRIIpolypeptide. Likewise, mutagenesis can give rise to variants which haveserum half-lives dramatically different than the corresponding wild-typeTβRII polypeptide. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or otherprocesses which result in destruction of, or otherwise elimination orinactivation of, a native TβRII polypeptide. Such variants, and thegenes which encode them, can be utilized to alter TβRII polypeptidelevels by modulating the half-life of the TβRII polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and can allow tighter control of recombinant TβRII polypeptidelevels within the patient. In an Fc fusion protein, mutations may bemade in the linker (if any) and/or the Fc portion to alter the half-lifeof the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential TβRII polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential TβRIIpolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential TβRII polypeptidevariants can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then be ligatedinto an appropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakuraet al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, TβRII polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of TβRII polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of TβRII polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include TβRII ligand binding assays and ligand-mediated cellsignaling assays.

In certain embodiments, the TβRII polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the TβRII polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, pegylation (polyethyleneglycol) and acylation. As a result, the modified TβRII polypeptides maycontain non-amino acid elements, such as polyethylene glycols, lipids,mono- or poly-saccharides, and phosphates. Effects of such non-aminoacid elements on the functionality of a TβRII polypeptide may be testedas described herein for other TβRII polypeptide variants. When a TβRIIpolypeptide is produced in cells by cleaving a nascent form of the TβRIIpolypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the TβRII polypeptides.

In certain aspects, functional variants or modified forms of the TβRIIpolypeptides include fusion proteins having at least a portion of theTβRII polypeptides and one or more fusion domains. Well-known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the TβRII polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, a TβRII polypeptide isfused with a domain that stabilizes the TβRII polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains.

As specific examples, the present disclosure provides fusion proteinscomprising variants of TβRII polypeptides fused to one of three Fcdomain sequences (e.g., SEQ ID NOs: 19, 20, and 21). Optionally, the Fcdomain has one or more mutations at residues such as Asp-265, Lys-322,and Asn-434 (numbered in accordance with the corresponding full-lengthIgG). In certain cases, the mutant Fc domain having one or more of thesemutations (e.g., Asp-265 mutation) has reduced ability of binding to theFcγ receptor relative to a wildtype Fc domain. In other cases, themutant Fc domain having one or more of these mutations (e.g., Asn-434mutation) has increased ability of binding to the MHC class I-relatedFc-receptor (FcRN) relative to a wildtype Fc domain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a TβRII polypeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to a TβRII polypeptide. The TβRII polypeptidedomain and the heterologous domain need not be adjacent in a fusionprotein, and additional domains or amino acid sequences may be includedC- or N-terminal to either domain or between the domains.

As used herein, the term “immunoglobulin Fc domain” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) andIgM (Igμ), may be used. The choice of appropriate immunoglobulin heavychain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087and 5,726,044. The choice of particular immunoglobulin heavy chainconstant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH3 domain of Fcgamma or the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613).

In certain embodiments, the present disclosure makes available isolatedand/or purified forms of the TβRII polypeptides, which are isolatedfrom, or otherwise substantially free of (e.g., at least 80%, 90%, 95%,96%, 97%, 98%, or 99% free of), other proteins and/or other TβRIIpolypeptide species. TβRII polypeptides will generally be produced byexpression from recombinant nucleic acids.

In certain embodiments, the disclosure includes nucleic acids encodingsoluble TβRII polypeptides comprising the coding sequence for anextracellular portion of a TβRII protein. In further embodiments, thisdisclosure also pertains to a host cell comprising such nucleic acids.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing the TβRIIpolypeptides.

3. Nucleic Acids Encoding TβRII Polypeptides

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the TβRII polypeptides, includingfragments, functional variants and fusion proteins disclosed herein. SEQID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 encode variants ofTβRII extracellular domain fused to an IgG2 Fc or an N-terminallytruncated IgG1 Fc domain. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids may be used, for example, in methods formaking TβRII polypeptides or as direct therapeutic agents (e.g., in anantisense, RNAi or gene therapy approach).

In certain aspects, the subject nucleic acids encoding TβRIIpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.Variant nucleotide sequences include sequences that differ by one ormore nucleotide substitutions, additions or deletions, such as allelicvariants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38,40, 42, and 44. One of ordinary skill in the art will appreciate thatnucleic acid sequences complementary to SEQ ID NOs: 26, 28, 30, 32, 34,36, 38, 40, 42, and 44, and variants of SEQ ID NOs: 26, 28, 30, 32, 34,36, 38, 40, 42, and 44 are also within the scope of this disclosure. Infurther embodiments, the nucleic acid sequences of the disclosure can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences designated in SEQ ID NOs: 26, 28, 30, 32, 34,36, 38, 40, 42, and 44 complement sequences of SEQ ID NOs: 26, 28, 30,32, 34, 36, 38, 40, 42, and 44, or fragments thereof. As discussedabove, one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In some embodiments, the disclosure provides nucleic acids whichhybridize under low stringency conditions of 6×SSC at room temperaturefollowed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 due todegeneracy in the genetic code are also within the scope of thedisclosure. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this disclosure.

It will be appreciated by one of skill in the art that correspondingvariants based on the long isoform of TβRII will include nucleotidesequences encoding the 25-amino acid insertion along with a conservativeVal-Ile substitution at the flanking position C-terminal to theinsertion. It will also be appreciated that corresponding variants basedon either the long (A) or short (B) isoforms of TβRII will includevariant nucleotide sequences comprising an insertion of 108 nucleotides,encoding a 36-amino-acid insertion (SEQ ID NO: 18), at the same locationdescribed for naturally occurring TβRII isoform C (see Exemplification).

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a TβRII polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the TβRII polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a TβRII polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant TβRII polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors(such as the ß-gal containing pBlueBac III).

In certain embodiments, a vector will be designed for production of thesubject TβRII polypeptides in CHO cells, such as a Pcmv-Script vector(Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad,Calif.) and pCI-neo vectors (Promega, Madison, Wis.). In a preferredembodiment, a vector will be designed for production of the subjectTβRII polypeptides in HEK-293 cells. As will be apparent, the subjectgene constructs can be used to cause expression of the subject TβRIIpolypeptides in cells propagated in culture, e.g., to produce proteins,including fusion proteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NOs: 26, 28,30, 32, 34, 36, 38, 40, 42, or 44) for one or more of the subject TβRIIpolypeptides. The host cell may be any prokaryotic or eukaryotic cell.For example, a TβRII polypeptide disclosed herein may be expressed inbacterial cells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject TβRII polypeptides. For example, a host celltransfected with an expression vector encoding a TβRII polypeptide canbe cultured under appropriate conditions to allow expression of theTβRII polypeptide to occur. The TβRII polypeptide may be secreted andisolated from a mixture of cells and medium containing the TβRIIpolypeptide. Alternatively, the TβRII polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, and media.Suitable media for cell culture are well known in the art. The subjectTβRII polypeptides can be isolated from cell culture medium, host cells,or both, using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, immunoaffinity purification withantibodies specific for particular epitopes of the TβRII polypeptidesand affinity purification with an agent that binds to a domain fused tothe TβRII polypeptide (e.g., a protein A column may be used to purify anTβRII-Fc fusion). In a preferred embodiment, the TβRII polypeptide is afusion protein containing a domain which facilitates its purification.As an example, purification may be achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant TβRIIpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified TβRII polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

Examples of categories of nucleic acid compounds that are antagonists ofTβRII, TGFβ1, TGFβ3 and GDF15 include antisense nucleic acids, RNAiconstructs and catalytic nucleic acid constructs. A nucleic acidcompound may be single or double stranded. A double stranded compoundmay also include regions of overhang or non-complementarity, where oneor the other of the strands is single-stranded. A single-strandedcompound may include regions of self-complementarity, meaning that thecompound forms a so-called “hairpin” or “stem-loop” structure, with aregion of double helical structure. A nucleic acid compound may comprisea nucleotide sequence that is complementary to a region consisting of nomore than 1000, no more than 500, no more than 250, no more than 100 orno more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-lengthTβRII nucleic acid sequence or ligand nucleic acid sequence. The regionof complementarity will preferably be at least 8 nucleotides, andoptionally at least 10 or at least 15 nucleotides, such as between 15and 25 nucleotides. A region of complementarity may fall within anintron, a coding sequence, or a noncoding sequence of the targettranscript, such as the coding sequence portion. Generally, a nucleicacid compound will have a length of about 8 to about 500 nucleotides orbase pairs in length, such as about 14 to about 50 nucleotides. Anucleic acid may be a DNA (particularly for use as an antisense), RNA,or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA,as well as modified forms that cannot readily be classified as eitherDNA or RNA. Likewise, a double-stranded compound may be DNA:DNA, DNA:RNAor RNA:RNA, and any one strand may also include a mixture of DNA andRNA, as well as modified forms that cannot readily be classified aseither DNA or RNA. A nucleic acid compound may include any of a varietyof modifications, including one or modifications to the backbone (thesugar-phosphate portion in a natural nucleic acid, includinginternucleotide linkages) or the base portion (the purine or pyrimidineportion of a natural nucleic acid). An antisense nucleic acid compoundwill preferably have a length of about 15 to about 30 nucleotides andwill often contain one or more modifications to improve characteristicssuch as stability in the serum, in a cell or in a place where thecompound is likely to be delivered, such as the stomach in the case oforally delivered compounds and the lung for inhaled compounds. In thecase of an RNAi construct, the strand complementary to the targettranscript will generally be RNA or modifications thereof. The otherstrand may be RNA, DNA, or any other variation. The duplex portion ofdouble-stranded or single-stranded “hairpin” RNAi construct willpreferably have a length of 18 to 40 nucleotides in length andoptionally about 21 to 23 nucleotides in length, so long as it serves asa Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymesor DNA enzymes and may also contain modified forms. Nucleic acidcompounds may inhibit expression of the target by about 50%, 75%, 90%,or more when contacted with cells under physiological conditions and ata concentration where a nonsense or sense control has little or noeffect. Preferred concentrations for testing the effect of nucleic acidcompounds are 1, 5 and 10 micromolar. Nucleic acid compounds may also betested for effects on, for example, angiogenesis.

4. Alterations in Fc-Fusion Proteins

The application further provides TβRII-Fc fusion proteins withengineered or variant Fc regions. Such antibodies and Fc fusion proteinsmay be useful, for example, in modulating effector functions, such as,antigen-dependent cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). Additionally, the modifications may improve thestability of the antibodies and Fc fusion proteins. Amino acid sequencevariants of the antibodies and Fc fusion proteins are prepared byintroducing appropriate nucleotide changes into the DNA, or by peptidesynthesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibodies and Fc fusion proteins disclosed herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibodies and Fc fusion proteins,such as changing the number or position of glycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may beproduced by introducing changes in the amino acid sequence, including,but are not limited to, the Ala-Ala mutation described by Bluestone etal. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 CellImmunol 200; 16-26). Thus, in certain embodiments, antibodies and Fcfusion proteins of the disclosure with mutations within the constantregion including the Ala-Ala mutation may be used to reduce or abolisheffector function. According to these embodiments, antibodies and Fcfusion proteins may comprise a mutation to an alanine at position 234 ora mutation to an alanine at position 235, or a combination thereof. Inone embodiment, the antibody or Fc fusion protein comprises an IgG4framework, wherein the Ala-Ala mutation would describe a mutation(s)from phenylalanine to alanine at position 234 and/or a mutation fromleucine to alanine at position 235. In another embodiment, the antibodyor Fc fusion protein comprises an IgG1 framework, wherein the Ala-Alamutation would describe a mutation(s) from leucine to alanine atposition 234 and/or a mutation from leucine to alanine at position 235.The antibody or Fc fusion protein may alternatively or additionallycarry other mutations, including the point mutation K322A in the CH2domain (Hezareh et al. 2001 J Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may bemodified to either enhance or inhibit complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region (see,e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved or reduced internalizationcapability and/or increased or decreased complement-mediated cellkilling. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes,B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature322: 738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351.

5. GDF15-TβRII Signaling

The present disclosure relates in part to the discovery that the TGFβtype II receptor (TβRII) binds to GDF15 with high affinity. Heretofore,GDF15 has not been shown biochemically to bind or interact directly witha receptor. Inadequate or inappropriate ligand purification could be apotential reason for the inactivity of commercially available GDF15.Exemplary GDF15 polypeptides demonstrating a TβRII binding activity andmethods of making and purifying such polypeptides are disclosed herein.Sequences of native precursor GDF15 proteins and nucleotides from humanand mouse are set forth in FIGS. 1-4. Mature human GDF15 extends fromresidues 197 to 308 of SEQ ID NO: 1. Similarly, mature mouse GDF15extends from residues 192 to 303 of SEQ ID NO: 3. In certainembodiments, the present disclosure makes available isolated and/orpurified forms of the GDF15 polypeptides or fragments thereof, which areisolated from, or otherwise substantially free of (e.g., at least 80%,90%, 95%, 96%, 97%, 98%, or 99% free of), other proteins and/or otherGDF15 polypeptide species. The GDF15 polypeptides of the disclosure bindto TβRII with high affinity. Binding may be assessed using purifiedproteins in solution or in a surface plasmon resonance system, such as aBiacore™ system. The GDF15 polypeptides will have an affinity (adissociation constant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M or less forTβRII polypeptides. Preferably, the GDF15 polypeptides of the disclosureare isolated and purified according to methods described herein. GDF15polypeptides will generally be produced by expression from recombinantnucleic acids.

GDF15 polypeptides may include a polypeptide consisting of, orcomprising, an amino acid sequence at least 80% identical, andoptionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the GDF15 polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3, or afunctional fragment thereof. GDF15 polypeptides may include apolypeptide consisting of, or comprising, an amino acid sequence atleast 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to the GDF15 polypeptide comprising residues197 to 308 of SEQ ID NO: 1 or residues 192 to 303 of SEQ ID NO: 3, or afunctional fragment thereof. The unprocessed GDF15 polypeptide mayeither include or exclude any signal sequence, as well as any sequenceN-terminal to the signal sequence. A GDF15 polypeptide may includevariants of SEQ ID NO: 1 or SEQ ID NO: 3, or portions thereof,corresponding to 197 to 308 of SEQ ID NO: 1 or residues 192 to 303 ofSEQ ID NO: 3, respectively (including variants that comprise, forexample, no more than 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acidsubstitutions in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3),fragments thereof, and fusion proteins comprising any of the foregoing,but in each case preferably any of the foregoing GDF15 polypeptides willpossess substantial affinity for a TβRII polypeptide.

In certain embodiments, the GDF15 polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the GDF15 polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, pegylation (polyethyleneglycol) and acylation. As a result, the modified GDF15 polypeptides maycontain non-amino acid elements, such as polyethylene glycols, lipids,mono- or poly-saccharides, and phosphates. Effects of such non-aminoacid elements on the functionality of a GDF15 polypeptide may be testedas described herein for other GDF15 polypeptides. When a GDF15polypeptide is produced in cells by cleaving a nascent form of the GDF15polypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the GDF15 polypeptides.

In certain embodiments, the disclosure includes nucleic acids encodingprecursor and mature GDF15 polypeptides. In further embodiments, thisdisclosure also pertains to a host cell comprising such nucleic acids.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing the GDF15polypeptides.

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the GDF15 polypeptides, includingfragments, functional variants and fusion proteins disclosed herein. Thesubject nucleic acids may be single-stranded or double-stranded. Suchnucleic acids may be DNA or RNA molecules. These nucleic acids may beused, for example, in methods for making GDF15 polypeptides or as directtherapeutic agents (e.g., in an antisense, RNAi or gene therapyapproach).

In certain aspects, the subject nucleic acids encoding GDF15polypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 1 or SEQ ID NO: 3. Variant nucleotide sequencesinclude sequences that differ by one or more nucleotide substitutions,additions or deletions, such as allelic variants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. One ofordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 1 or SEQ ID NO: 3 and variants of SEQ ID NO:1 or SEQ ID NO: 3 are also within the scope of this disclosure. Infurther embodiments, the nucleic acid sequences of the disclosure can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences designated in SEQ ID NO: 1 or SEQ ID NO: 3,complement sequences of SEQ ID NO: 1 or SEQ ID NO: 3, or fragmentsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In some embodiments, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 1 or SEQ ID NO: 3 due to degeneracy in the genetic codeare also within the scope of the disclosure. For example, a number ofamino acids are designated by more than one triplet. Codons that specifythe same amino acid, or synonyms (for example, CAU and CAC are synonymsfor histidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a GDF15 polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the GDF15 polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a GDF15 polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant GDF15 polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors(such as the ß-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject TβRII polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.), pCI-neo vectors (Promega, Madison, Wis.) andUCOE™-derived vectors (Millipore). As will be apparent, the subject geneconstructs can be used to cause expression of the subject GDF15polypeptides in cells propagated in culture, e.g., to produce proteins,including fusion proteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 1 or SEQID NO:3) for one or more of the subject GDF15 polypeptides. The hostcell may be any prokaryotic or eukaryotic cell. For example, a GDF15polypeptide disclosed herein may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art. In a preferred embodiment, a GDF15 polypeptidedisclosed herein is expressed in CHO cells.

Accordingly, the present disclosure further pertains to methods ofproducing the subject GDF15 polypeptides. For example, a host celltransfected with an expression vector encoding a GDF15 polypeptide canbe cultured under appropriate conditions to allow expression of theGDF15 polypeptide to occur. The GDF15 polypeptide may be secreted andisolated from a mixture of cells and medium containing the GDF15polypeptide. Alternatively, the TβRII polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells and media.Suitable media for cell culture are well known in the art. The subjectGDF15 polypeptides can be isolated from cell culture medium, host cells,or both, using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, immunoaffinity purification withantibodies specific for particular epitopes of the GDF15 polypeptidesand affinity purification with an agent that binds to a domain fused tothe GDF15 polypeptide (e.g., a protein A column may be used to purify anGDF15-Fc fusion). In a preferred embodiment, the GDF15 polypeptide is afusion protein containing a domain which facilitates its purification.As an example, purification may be achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In a preferred embodiment, the subject GDF15 polypeptides are purifiedfrom culture media using a series of cation-exchange columnchromatography steps. Examples of the material used for the cationexchange column can be resins having substituents such as carboxymethyl(CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate(S). Examples of the material used for the cation exchange columnchromatography include SP Sepharose™ Fast Flow, Q Sepharose™ Fast Flow,DEAE Sepharose™ Fast Flow, Capto™5, Capto™ DEAE (GE Healthcare), SHyperCel™ (Pall), TOYOPEARL GigaCap S-650 (TOSOH) or weak cationexchangers such as carboxymethyl. SP Sepharose™ Fast Flow and QSepharose™ Fast Flow are preferred.

To begin purification, parameters of the conditioned media from hostcells stably expressing a GDF15 polypeptide, such as pH, ionic strength,and temperature may be adjusted if necessary. In some embodiments, achromatography column is flushed and equilibrated with one or moresolutions prior to contact with a polypeptide containing supernatant.Such solutions can include, for example, a buffer (e.g., Tris, MES,HEPES, histidine, phosphate or sodium acetate, e.g., between 1-500 mM,25-100 mM, 15-30 mM or 20 mM), and/or salt (e.g., NaCl, NaPO₄ sodiumacetate, or CaCl₂, e.g., between 0-2 M, 1-2 M or 500 mM-1M). The pH ofan equilibration solution generally ranges from 3.5-10 (e.g., between pH3.5-6, 4.0-5.5, 4.5-4.8 or 4.7). After contacting a column with apolypeptide containing fluid, the bound column can be washed. Washsolutions can include a buffer (e.g., Tris, MES, HEPES, histidine,phosphate, or sodium acetate, e.g., between 1-500 mM, 25-100 mM, 15-30mM or 20 mM), and/or salt (e.g., NaCl, NaPO₄, sodium acetate, or CaCl₂),e.g., between 0-2 M, 1-2 M, 100 mM-1M or 100 mM-500 mM), and/or anadditive (e.g. guanidine, urea, sucrose, arginine, or an argininederivative), and/or a solvent (e.g., ethanol, acetonitrile, orpolyethylene glycol). Wash solutions generally have a pH between 3.5 and10 (e.g., a pH between 4.5-8.0). Polypeptides can be eluted from acolumn using a step or gradient change in pH, salt type, saltconcentration, solvent type, solvent concentration, displacer type,displacer concentration, or a combination thereof. In general, to elutea polypeptide from a column, the medium is contacted with an elutionbuffer. In some embodiments, an elution buffer elution buffer contains abuffer (e.g., HEPES or Tris, e.g., 10-100 mM, 25-75 mM or 50 mM) and/orcontains a salt (e.g., NaCl or CaCl₂), e.g., 0-2 M, e.g., 10-100 mM). Insome embodiments, an elution buffer may contain glycine, acetic acid, orcitric acid (e.g., 20-250 mM, or 150 mM). An elution buffer may alsocontain acetic acid (e.g., 20 mM to about 50 mM), an additive (e.g.guanidine, urea, or sucrose, e.g., 1-10 M, 2-8 M or 6 M), and/or asolvent (e.g., ethanol, acetonitrile, polyethylene glycol, e.g., 1-10%solvent, e.g., 5% solvent). The pH of the elution buffer may range fromabout 5.0 to about 10.0. In some embodiments, pH can be changed (e.g.,gradually) to produce a gradient elution. In some embodiments, the pH ofthe elution buffer is about 8.0. In some embodiments, a series of columnchromatography steps are performed.

The data presented herein demonstrates that TβRII polypeptides act asantagonists of GDF15 signaling. Although soluble TβRII polypeptides, andparticularly T3RII-Fc, are preferred antagonists, other types of GDF15antagonists are expected to be useful, including anti-GDF15 antibodies,anti-TβRII antibodies, antisense, RNAi or ribozyme nucleic acids thatinhibit the production of GDF15 or TβRII and other inhibitors of GDF15or TβRII, particularly those that disrupt GDF15-TβRII binding.

An antibody that is specifically reactive with a GDF15 polypeptide andwhich either binds to GDF15 polypeptide so as to compete with itsbinding to TβRII polypeptide (binding competitively) or otherwiseinhibits GDF15-mediated signaling may be used as an antagonist of GDF15polypeptide activities. Likewise, an antibody that is specificallyreactive with a TβRII polypeptide and which disrupts GDF15 binding maybe used as an antagonist.

By using immunogens derived from a GDF15 polypeptide or a TβRIIpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the GDF15 polypeptide, an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion protein.Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of a GDF15 or TβRII polypeptide can be administeredin the presence of adjuvant. The progress of immunization can bemonitored by detection of antibody titers in plasma or serum. StandardELISA or other immunoassays can be used with the immunogen as antigen toassess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of aGDF15 polypeptide, antisera can be obtained and, if desired, polyclonalantibodies can be isolated from the serum. To produce monoclonalantibodies, antibody-producing cells (lymphocytes) can be harvested froman immunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique (originally developed by Kohler and Milstein, (1975)Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar etal., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a GDF15 polypeptide and monoclonal antibodiesisolated from a culture comprising such hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject polypeptide.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific, single-chain, chimeric, humanized and fully human moleculeshaving affinity for an TβRII or GDF15 polypeptide conferred by at leastone CDR region of the antibody. An antibody may further comprise a labelattached thereto and able to be detected (e.g., the label can be aradioisotope, fluorescent compound, enzyme or enzyme co-factor).

In certain embodiments, the antibody is a recombinant antibody, whichterm encompasses any antibody generated in part by techniques ofmolecular biology, including CDR-grafted or chimeric antibodies, humanor other antibodies assembled from library-selected antibody domains,single chain antibodies and single domain antibodies (e.g., human V_(H)proteins or camelid V_(HH) proteins). In certain embodiments, anantibody of the invention is a monoclonal antibody, and in certainembodiments, the invention makes available methods for generating novelantibodies. For example, a method for generating a monoclonal antibodythat binds specifically to a GDF15 polypeptide or TβRII polypeptide maycomprise administering to a mouse an amount of an immunogeniccomposition comprising the antigen polypeptide effective to stimulate adetectable immune response, obtaining antibody-producing cells (e.g.,cells from the spleen) from the mouse and fusing the antibody-producingcells with myeloma cells to obtain antibody-producing hybridomas, andtesting the antibody-producing hybridomas to identify a hybridoma thatproduces a monoclonal antibody that binds specifically to the antigen.Once obtained, a hybridoma can be propagated in a cell culture,optionally in culture conditions where the hybridoma-derived cellsproduce the monoclonal antibody that binds specifically to the antigen.The monoclonal antibody may be purified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., a GDF15 polypeptide) and other antigens that are not ofinterest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less. Given the highaffinity between GDF15 and TβRII, it is expected that a neutralizinganti-GDF15 or anti-TβRII antibody would generally have a dissociationconstant of 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are GDF15 or TβRIIantagonists include antisense nucleic acids, RNAi constructs andcatalytic nucleic acid constructs. A nucleic acid compound may besingle- or double-stranded. A double-stranded compound may also includeregions of overhang or non-complementarity, where one or the other ofthe strands is single-stranded. A single-stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100 or no morethan 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length GDF15nucleic acid sequence or TβRII nucleic acid sequence. The region ofcomplementarity will preferably be at least 8 nucleotides, andoptionally at least 10 or at least 15 nucleotides, such as between 15and 25 nucleotides. A region of complementarity may fall within anintron, a coding sequence or a noncoding sequence of the targettranscript, such as the coding sequence portion. Generally, a nucleicacid compound will have a length of about 8 to about 500 nucleotides orbase pairs in length, such as about 14 to about 50 nucleotides. Anucleic acid may be a DNA (particularly for use as an antisense), RNA orRNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, aswell as modified forms that cannot readily be classified as either DNAor RNA. Likewise, a double-stranded compound may be DNA:DNA, DNA:RNA orRNA:RNA, and any one strand may also include a mixture of DNA and RNA,as well as modified forms that cannot readily be classified as eitherDNA or RNA. A nucleic acid compound may include any of a variety ofmodifications, including one or modifications to the backbone (thesugar-phosphate portion in a natural nucleic acid, includinginternucleotide linkages) or the base portion (the purine or pyrimidineportion of a natural nucleic acid). An antisense nucleic acid compoundwill preferably have a length of about 15 to about 30 nucleotides andwill often contain one or more modifications to improve characteristicssuch as stability in the serum, in a cell or in a place where thecompound is likely to be delivered, such as the stomach in the case oforally delivered compounds and the lung for inhaled compounds. In thecase of an RNAi construct, the strand complementary to the targettranscript will generally be RNA or modifications thereof. The otherstrand may be RNA, DNA or any other variation. The duplex portion ofdouble-stranded or single-stranded “hairpin” RNAi construct willpreferably have a length of 18 to 40 nucleotides in length andoptionally about 21 to 23 nucleotides in length, so long as it serves asa Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymesor DNA enzymes and may also contain modified forms. Nucleic acidcompounds may inhibit expression of the target by about 50%, 75%, 90% ormore when contacted with cells under physiological conditions and at aconcentration where a nonsense or sense control has little or no effect.Preferred concentrations for testing the effect of nucleic acidcompounds are 1, 5 and 10 micromolar.

6. Screening Assays

In certain aspects, the present invention relates to the use of TβRIIpolypeptides (e.g., soluble TβRII polypeptides) and GDF15 polypeptidesto identify compounds (agents) which are agonist or antagonists of theGDF15-TβRII signaling pathway. Compounds identified through thisscreening can be tested to assess their ability to modulate GDF15signaling activity in vitro. Optionally, these compounds can further betested in animal models to assess their ability to modulate tissuegrowth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting GDF15 and TβRII polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb GDF15 or TβRII-mediated cellsignaling. In certain embodiments, the assay is carried out to screenand identify compounds that specifically inhibit or reduce binding of aTβRII polypeptide to GDF15. Alternatively, the assay can be used toidentify compounds that enhance binding of a TβRII polypeptide to GDF15.In a further embodiment, the compounds can be identified by theirability to interact with a GDF15 or TβRII polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a TβRIIpolypeptide and GDF15.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified TβRII polypeptide which is ordinarily capable of binding toGDF15. To the mixture of the compound and TβRII polypeptide is thenadded a composition containing a TβRII ligand. Detection andquantification of TβRII/GDF15 complexes provides a means for determiningthe compound's efficacy at inhibiting (or potentiating) complexformation between the TβRII polypeptide and GDF15. The efficacy of thecompound can be assessed by generating dose response curves from dataobtained using various concentrations of the test compound. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. For example, in a control assay, isolated and a purifiedGDF15 is added to a composition containing the TβRII polypeptide, andthe formation of TβRII/GDF15 complex is quantitated in the absence ofthe test compound. It will be understood that, in general, the order inwhich the reactants may be admixed can be varied, and can be admixedsimultaneously. Moreover, in place of purified proteins, cellularextracts and lysates may be used to render a suitable cell-free assaysystem.

Complex formation between the TβRII polypeptide and GDF15 may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled TβRIIpolypeptide or GDF15, by immunoassay, or by chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a TβRII polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a TβRII polypeptide andits binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between a TβRII polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with a TβRII or GDF15 polypeptide of the invention.The interaction between the compound and the TβRII or GDF15 polypeptidemay be covalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to a GDF15 orTβRII polypeptide. This may include a solid-phase or fluid-phase bindingevent. Alternatively, the gene encoding a GDF15 or TβRII polypeptide canbe transfected with a reporter system (e.g., β-galactosidase,luciferase, or green fluorescent protein) into a cell and screenedagainst the library preferably by a high-throughput screening or withindividual members of the library. Other mechanism-based binding assaysmay be used, for example, binding assays which detect changes in freeenergy. Binding assays can be performed with the target fixed to a well,bead or chip or captured by an immobilized antibody or resolved bycapillary electrophoresis. The bound compounds may be detected usuallyusing colorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) GDF15-mediated cellsignaling. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate GDF15 signaling. Various methods known in the art can beutilized for this purpose.

7. Exemplary Therapeutic Uses

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician and the intended result ofadministration of the therapeutic agent.

The disclosure provides methods of treating or preventing a disease orcondition associated with a TGFβ superfamily member by administering toa subject an effective amount of a TβRII polypeptide, including aTβRII-Fc fusion protein or nucleic acid antagonists (e.g., antisense orsiRNA) of the foregoing, hereafter collectively referred to as“therapeutic agents”. In some embodiments the disease or condition isassociated with dysregulated GDF15, TGFβ1 or TGFβ3 signaling. Alsoprovided are methods and compositions for treating certaincardiovascular or vascular disorders. In addition, the disclosureprovides methods and compositions for treating or preventing cancer. Inaddition, the disclosure provides methods and compositions for treatingor preventing fibrotic disorders and conditions.

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing chronic vascular or cardiovasculardiseases. Exemplary disorders of this kind include, but are not limitedto, heart disease (including myocardial disease, myocardial infarct,angina pectoris, and heart valve disease); renal disease (includingchronic glomerular inflammation, diabetic renal failure, andlupus-related renal inflammation); disorders associated withatherosclerosis or other types of arteriosclerosis (including stroke,cerebral hemorrhage, subarachnoid hemorrhage, angina pectoris, and renalarteriosclerosis); thrombotic disorders (including cerebral thrombosis,thrombotic intestinal necrosis); complications of diabetes (includingdiabetes-related retinal disease, cataracts, diabetes-related renaldisease, diabetes-related neuropathology, diabetes-related gangrene, anddiabetes-related chronic infection); vascular inflammatory disorders(systemic lupus erythematosus, joint rheumatism, joint arterialinflammation, large-cell arterial inflammation, Kawasaki disease,Takayasu arteritis, Churg-Strauss syndrome, and Henoch-Schoenleinpurpura); diabetic vasculopathies; and cardiac disorders such ascongenital heart disease, cardiomyopathy (e.g., dilated, hypertrophic,restrictive cardiomyopathy), and congestive heart failure. Exemplarydisorders further include, but are not limited to, hereditaryhemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome,familial thoracic aortic aneurysm syndrome, arterial tortuositysyndrome, pre-eclampsia, and restenosis.

The TβRII polypeptide can be administered to the subject alone, or incombination with one or more agents or therapeutic modalities, e.g.,therapeutic agents, which are useful for treating TGFβ associatedcardiovascular disorders and/or conditions. In certain embodiments, thesecond agent or therapeutic modality is chosen from one or more of:angioplasty, beta blockers, anti-hypertensives, cardiotonics,anti-thrombotics, vasodilators, hormone antagonists, endothelinantagonists, calcium channel blockers, phosphodiesterase inhibitors,angiotensin type 2 antagonists and/or cytokine blockers/inhibitors

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing a cancer (tumor). The terms“cancer” and “cancerous” refer to or describe, the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth/proliferation. Examples of cancer, or neoplastic disorders,include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, stomach cancer,intestinal cancer, skin cancer, bone cancer, gastric cancer, melanoma,and various types of head and neck cancer, including squamous cell headand neck cancer. Other examples of neoplastic disorders and relatedconditions include esophageal carcinomas, thecomas, arrhenoblastomas,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, Wilm'stumor, renal cell carcinoma, prostate carcinoma, abnormal vascularproliferation associated with phakomatoses, and Meigs' syndrome. Acancer that is particularly amenable to treatment with the therapeuticagents described herein may be characterized by one or more of thefollowing: the cancer has elevated TβRII levels detectable in the tumoror the serum, increased GDF15, TGFβ1 or TGFβ3 expression levels orbiological activity, is metastatic or at risk of becoming metastatic, orany combination thereof.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject methods of the disclosure can beused alone. Alternatively, the subject methods may be used incombination with other conventional anti-cancer therapeutic approachesdirected to treatment or prevention of proliferative disorders (e.g.,tumor). For example, such methods can be used in prophylactic cancerprevention, prevention of cancer recurrence and metastases aftersurgery, and as an adjuvant of other conventional cancer therapy. Thepresent disclosure recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of a subjectpolypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic or anti-cancer activities. These compounds have beenused as pharmaceutical agents in chemotherapy to shrink solid tumors,prevent metastases and further growth, or decrease the number ofmalignant cells in leukemic or bone marrow malignancies. Althoughchemotherapy has been effective in treating various types ofmalignancies, many anti-neoplastic compounds induce undesirable sideeffects. It has been shown that when two or more different treatmentsare combined, the treatments may work synergistically and allowreduction of dosage of each of the treatments, thereby reducing thedetrimental side effects exerted by each compound at higher dosages. Inother instances, malignancies that are refractory to a treatment mayrespond to a combination therapy of two or more different treatments.

When a therapeutic agent disclosed herein is administered in combinationwith another conventional anti-neoplastic agent, either concomitantly orsequentially, such therapeutic agent may enhance the therapeutic effectof the anti-neoplastic agent or overcome cellular resistance to suchanti-neoplastic agent. This allows decrease of dosage of ananti-neoplastic agent, thereby reducing the undesirable side effects, orrestores the effectiveness of an anti-neoplastic agent in resistantcells.

According to the present disclosure, the polypeptide therapeutic agentsdescribed herein may be used in combination with other compositions andprocedures for the treatment of diseases. For example, a tumor may betreated conventionally with surgery, radiation or chemotherapy combinedwith the TβRII polypeptide, and then the TβRII polypeptide may besubsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize any residual primary tumor.

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with a TβRII polypeptide include other cancertherapies: e.g., surgery, cytotoxic agents, radiological treatmentsinvolving irradiation or administration of radioactive substances,chemotherapeutic agents, anti-hormonal agents, growth inhibitory agents,anti-neoplastic compositions, and treatment with anti-cancer agentslisted herein and known in the art, or combinations thereof.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, l¹³¹, l¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cyclophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as folinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elfornithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhóne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LYl 17018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In addition,such definition of chemotherapeutic agents includes bisphosphonates suchas clodronate (for example, BONEFOS® or OSTAC®), DIDROC AL® etidronate,NE-58095, ZOMET A® zoledronic acid/zoledronate, FOSAMAX® alendronate,AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in aberrant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as THERATOPE®vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine,LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1inhibitor; ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dualtyrosine kinase small-molecule inhibitor also known as GW572016); andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce Gl arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest Gl also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

In still other embodiments, TβRII polypeptides may be useful in thetreatment or prevention of fibrosis. As used herein, the term “fibrosis”refers to the aberrant formation or development of excess fibrousconnective tissue by cells in an organ or tissue. Although processesrelated to fibrosis can occur as part of normal tissue formation orrepair, dysregulation of these processes can lead to altered cellularcomposition and excess connective tissue deposition that progressivelyimpairs to tissue or organ function. The formation of fibrous tissue canresult from a reparative or reactive process. Fibrotic disorders orconditions include, but are not limited to, fibroproliferative disordersassociated with vascular diseases, such as cardiac disease, cerebraldisease, and peripheral vascular disease, as well as tissues and organsystems including the heart, skin, kidney, peritoneum, gut, and liver(as disclosed in, e.g., Wynn, 2004, Nat Rev 4:583-594, incorporatedherein by reference). Exemplary disorders that can be treated include,but are not limited to, renal fibrosis, including nephropathiesassociated with injury/fibrosis, e.g., chronic nephropathies associatedwith diabetes (e.g., diabetic nephropathy), lupus, scleroderma,glomerular nephritis, focal segmental glomerular sclerosis, and IgAnephropathy; gut fibrosis, e.g., scleroderma, and radiation-induced gutfibrosis; liver fibrosis, e.g., cirrhosis, alcohol-induced liverfibrosis, biliary duct injury, primary biliary cirrhosis, infection orviral-induced liver fibrosis, congenital hepatic fibrosis and autoimmunehepatitis; and other fibrotic conditions, such as cystic fibrosis,endomyocardial fibrosis, mediastinal fibrosis, sarcoidosis, scleroderma,spinal cord injury/fibrosis, myelofibrosis, vascular restenosis,atherosclerosis, injection fibrosis (which can occur as a complicationof intramuscular injections, especially in children), endomyocardialfibrosis, retroperitoneal fibrosis, and nephrogenic systemic fibrosis.

As used herein, the terms “fibrotic disorder”, “fibrotic condition,” and“fibrotic disease,” are used interchangeably to refer to a disorder,condition or disease characterized by fibrosis. Examples of fibroticdisorders include, but are not limited to sclerotic disorders (e.g.,scleroderma, atherosclerosis, diffuse systemic sclerosis), vascularfibrosis, pancreatic fibrosis, liver fibrosis (e.g., cirrhosis), renalfibrosis, musculoskeletal fibrosis, cardiac fibrosis (e.g.,endomyocardial fibrosis, idiopathic myocardiopathy), skin fibrosis(e.g., scleroderma, post-traumatic, operative cutaneous scarring,keloids and cutaneous keloid formation), eye fibrosis (e.g., glaucoma,sclerosis of the eyes, conjunctival and corneal scarring, andpterygium), myelofibrosis, progressive systemic sclerosis (PSS), chronicgraft-versus-host disease, Peyronie's disease, post-cystoscopic urethralstenosis, idiopathic and pharmacologically induced retroperitonealfibrosis, mediastinal fibrosis, proliferative fibrosis, neoplasticfibrosis, Dupuytren's disease, strictures, neural scarring, dermalscarring and radiation induced fibrosis.

As used herein, inhibition of the fibrotic response of a cell, includes,but is not limited to the inhibition of the fibrotic response of one ormore cells within the liver (or liver tissue); one or more cells withinthe kidney (or renal tissue); one or more cells within muscle tissue;one or more cells within the heart (or cardiac tissue); one or morecells within the pancreas; one or more cells within the skin; one ormore cells within the bone, one or more cells within the vasculature,one or more stem cells, or one or more cells within the eye.

The present invention contemplates the use of TβRII polypeptides incombination with one or more other therapeutic modalities. Thus, inaddition to the use of TβRII polypeptides, one may also administer tothe subject one or more “standard” therapies for treating fibroticdisorders. For example, the TβRII polypeptides can be administered incombination with (i.e., together with) cytotoxins, immunosuppressiveagents, radiotoxic agents, and/or therapeutic antibodies. Particularco-therapeutics contemplated by the present invention include, but arenot limited to, steroids (e.g., corticosteroids, such as Prednisone),immune-suppressing and/or anti-inflammatory agents (e.g.,gamma-interferon, cyclophosphamide, azathioprine, methotrexate,penicillamine, cyclosporine, colchicine, antithymocyte globulin,mycophenolate mofetil, and hydroxychloroquine), cytotoxic drugs, calciumchannel blockers (e.g., nifedipine), angiotensin converting enzymeinhibitors (ACE) inhibitors, para-aminobenzoic acid (PABA), dimethylsulfoxide, transforming growth factor beta (TGFβ) inhibitors,interleukin-5 (IL-5) inhibitors, and pan caspase inhibitors.

Additional anti-fibrotic agents that may be used in combination withTβRII polypeptides include, but are not limited to, lectins (asdescribed in, for example, U.S. Pat. No. 7,026,283, the entire contentsof which is incorporated herein by reference), as well as theanti-fibrotic agents described by Wynn et al (2007, J Clin Invest117:524-529, the entire contents of which is incorporated herein byreference). For example, additional anti-fibrotic agents and therapiesinclude, but are not limited to, variousanti-inflammatory/immunosuppressive/cytotoxic drugs (includingcolchicine, azathioprine, cyclophosphamide, prednisone, thalidomide,pentoxifylline and theophylline), TGFβ signaling modifiers (includingrelaxin, SMAD7, HGF, and BMP7, as well as TGFβ1, TβRI, TβRII, EGR-I, andCTGF inhibitors), cytokine and cytokine receptor antagonists (inhibitorsof IL-1β, IL-5, IL-6, IL-13, IL-21, IL-4R, IL-13Rα1, GM-CSF, TNF-α,oncostatin M, WlSP-I, and PDGFs), cytokines and chemokincs (IFN-γ,IFN-α/β, IL-12, IL-10, HGF, CXCL10, and CXCL11), chemokine antagonists(inhibitors of CXCL1, CXCL2, CXCL12, CCL2, CCL3, CCL6, CCL17, andCCL18), chemokine receptor antagonists (inhibitors of CCR2, CCR3, CCR5,CCR7, CXCR2, and CXCR4), TLR antagonists (inhibitors of TLR3, TLR4, andTLR9), angiogenesis antagonists (VEGF-specific antibodies and adenosinedeaminase replacement therapy), antihypertensive drugs (beta blockersand inhibitors of ANG 11, ACE, and aldosterone), vasoactive substances(ET-1 receptor antagonists and bosetan), inhibitors of the enzymes thatsynthesize and process collagen (inhibitors of prolyl hydroxylase), Bcell antagonists (rituximab), integrin/adhesion molecule antagonists(molecules that block αlβl and αvβ6 integrins, as well as inhibitors ofintegrin-linked kinase, and antibodies specific for ICAM-I and VCAM-I),proapoptotic drugs that target myofibroblasts, MMP inhibitors(inhibitors of MMP2, MMP9, and MMP12), and T1MP inhibitors (antibodiesspecific for TIMP-1).

The TβRII polypeptide and the co-therapeutic agent or co-therapy can beadministered in the same formulation or separately. In the case ofseparate administration, the TβRII polypeptide can be administeredbefore, after, or concurrently with the co-therapeutic or co-therapy.One agent may precede or follow administration of the other agent byintervals ranging from minutes to weeks. In embodiments where two ormore different kinds of therapeutic agents are applied separately to asubject, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that thesedifferent kinds of agents would still be able to exert an advantageouslycombined effect on the target tissues or cells.

8. Pharmaceutical Compositions

The therapeutic agents described herein (e.g., TβRII polypeptides) maybe formulated into pharmaceutical compositions. Pharmaceuticalcompositions for use in accordance with the present disclosure may beformulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. Such formulations will generally besubstantially pyrogen-free, in compliance with most regulatoryrequirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the TβRII signalingantagonists which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., TβRII polypeptides) in the methodsdisclosed herein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more TβRII polypeptides incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, compositionsof the present invention may include a matrix capable of delivering oneor more therapeutic compounds (e.g., TβRII polypeptides) to a targettissue site, providing a structure for the developing tissue andoptimally capable of being resorbed into the body. For example, thematrix may provide slow release of the TβRII polypeptides. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalcium phosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornonaqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof, and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., TβRII polypeptides).The various factors include, but are not limited to, the patient's age,sex, and diet, the severity disease, time of administration, and otherclinical factors. Optionally, the dosage may vary with the type ofmatrix used in the reconstitution and the types of compounds in thecomposition. The addition of other known growth factors to the finalcomposition, may also affect the dosage. Progress can be monitored byperiodic assessment of bone growth and/or repair, for example, X-rays(including DEXA), histomorphometric determinations, and tetracyclinelabeling.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of TβRII polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the TβRIIpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of TβRII polynucleotide sequences can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Preferred for therapeutic delivery of TβRIIpolynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the TβRII polynucleotide. In a preferred embodiment,the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for TβRII polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1. Generation of Bioactive GDF15

GDF15 (also known as macrophage-inhibitory cytokine-1) has not beenshown biochemically to bind or interact directly with any receptor.Applicants first tried without success to identify a native receptorwith high-affinity binding to GDF15 using commercially available humanGDF15 (R&D Systems) produced in mammalian CHO cells. Like other ligandsin the TGFβ superfamily, which contain a characteristic cysteine knotmotif, mature GDF15 is synthesized with a larger prodomain (Harrison etal., Growth Factors 29:174, 2011; Shi et al., Nature 474:343, 2011) thatis removed through cleavage by a furin-like protease at the canonicalRXXR site to generate mature dimeric GDF15. Since inadequate orinappropriate ligand purification could be a potential reason forinactivity of commercially available GDF15, Applicants tested differentpurification procedures for GDF15.

Stable Expression of GDF15 in CHO Cells

Applicants used CHO cells to express human GDF15 (hGDF15) and murineGDF15 (mGDF15) for further studies. The amino acid sequence of nativeprecursor for hGDF15 is shown in FIG. 1, and a corresponding nucleotidesequence (with a silent, single nucleotide substitution compared to thenative sequence) is shown in FIG. 2. The native amino acid andnucleotide sequences for mGDF15 precursor are shown in FIGS. 3 and 4,respectively. For expression in CHO cells, UCOE™-based constructsencoding human or murine GDF15 precursor were stably transfected into aCHO-PACE cell line. Clones were selected in methotrexate levels of 10nM, 20 nM, and 50 nM, and any clones that formed colonies (one or twoper methotrexate concentration) were then pooled. No gene amplificationwas performed since it is difficult to amplify UCOE™ pools whilemaintaining stability of expression. Instead of dilution cloning,high-expressing pools were identified and used for generating hGDF15 andmGDF15.

Purification of Human GDF15

To begin purification, conditioned media from CHO cells stablyexpressing hGDF15 was adjusted to pH 4.7 with acetic acid. Afterincubation of media for 10 min at ambient temperature, precipitate wasremoved by centrifugation. Supernatant was filtered with a 0.8 mdisposable filter. An SP Sepharose™ Fast Flow column (GE Healthcare) wasequilibrated with buffers A (20 mM sodium acetate, pH 4.7) and B (20 mMsodium acetate, 1M NaCl, pH 4.7). Loading was performed at 100 cm/hr.The column was washed with 20% B (200 mM NaCl) until no more proteineluted from the column and then washed back to 0% B to remove anyresidual salt. Protein was eluted with 50 mM Tris, 6M urea, pH 8.0(Tris+urea pool) until no more protein eluted from the column, followedby elution with 50 mM Tris, 6M urea, 1M NaCl, pH 8.0 (Tris+urea+saltpool). Each pool was dialyzed in 50 mM 4-morpholineethanesulfonic acid(MES, pH 6.5) overnight at 4° C.

GDF15 found in the Tris+urea+salt pool was degraded based on Westernblot analysis, so this pool was discarded. The Tris+urea pool was loadedon a Q Sepharose™ Fast Flow column (GE Healthcare) previouslyequilibrated with buffers A (50 mM MES, pH 6.5) and B (50 mM MES, 1MNaCl, pH 6.5). The flow-through was collected, and the column was washedwith 10% B (100 mM NaCl), followed by a 10-50% B gradient (100-500 mMNaCl) over five column volumes at 120 cm/hr. After evaluation of theflow-through and wash fractions by Western blot, protein was foundmainly in the flow-through. The flow-through was injected on areverse-phase preparative C4 column (Vydac) attached to a HPLC, withbuffers A (water/0.1% TFA) and B (acetonitrile/0.1% TFA). A 25-40% Bgradient over 1 h at 4.5 mL/min produced the best resolution. Collectedfractions were evaluated by SDS-PAGE gel (Sypro Ruby) and Western blotto select those for concentration in a centrifugal evaporator.

Purification of Murine GDF15

The pH of the conditioned media was adjusted to pH 4.7 with acetic acid.After incubation of media for 10 min at ambient temperature, precipitatewas removed by centrifugation. Supernatant was filtered with a 0.8 mdisposable filter. An SP Sepharose™ Fast Flow column (GE Healthcare) wasequilibrated with buffers A (20 mM sodium acetate, pH 4.7) and B (20 mMsodium acetate, 1M NaCl, pH 4.7). Loading was performed at 100-150cm/hr, and the column was washed with buffer A until no more proteineluted from the column. A wash was performed at 60% B (600 mM NaCl) for3-4 column volumes, followed by elution with 100% B (1M NaCl) for 3-4column volumes. Elution continued with 50 mM Tris, 6M urea, pH 8.0, toremove any protein still bound to the resin.

Non-reduced samples of SP-column fractions were analyzed by Westernblot. Although most protein was found in the Tris-eluted fractions,previous experiments have indicated that mGDF15 found in these fractionsis essentially inactive, so it was not used for further purification.Instead, purification was continued with protein found in the 100% Belution (salt-elution pool). This pool was injected on a reverse-phasepreparative C4 column (Vydac) attached to a HPLC. Buffer A waswater/0.1% TFA and buffer B was acetonitrile/0.1% TFA. Protein waseluted with a 25-40% B gradient over 1 h at 4.5 mL/min. After evaluationof the reverse-phase column fractions by SDS-PAGE gel (Sypro Ruby) andWestern blot, the fractions containing pure mGDF15 were pooled andconcentrated in a centrifugal evaporator.

The identities of hGDF15 and mGDF15 were each confirmed by N-terminalsequencing. Both types of purified GDF15 stimulated SMAD2/3phosphorylation in two different cell lines, thereby providingconfirmation of ligand activity.

Example 2. Identification of a TGFβ Superfamily Receptor withHigh-Affinity Binding to GDF15

Once active GDF15 protein was obtained, receptor-Fc fusion proteinscomprising TGFβ superfamily receptors were screened for binding to humanor murine GDF15 that was generated and purified as described inExample 1. These fusion proteins incorporated an IgG1 Fc domain and wereeither purchased from R&D Systems or generated in-house. Among the fivetype II receptors (TGFβ receptor type II, activin receptor type IIA,activin receptor type IIB, BMP receptor type II, and MIS receptor typeII), only TGFβ receptor type II (TβRII) exhibited detectable binding toGDF15 (k_(a)=2.92×10⁵ M⁻¹ s⁻¹; k_(d)=0.001 s⁻¹), as determined bysurface plasmon resonance with captured receptor-Fc fusion proteins.hGDF15 bound to captured hTβRII-Fc at 37° C. with an equilibriumdissociation constant (K_(D)) of 9.56 nM. None of the seven type Ireceptors (ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7) displayeddetectable binding to GDF15 (mGDF15 at 20 nM or 200 nM).

Human TβRII occurs naturally in at least two isoforms—A (long) and B(short)-generated by alternative splicing in the extracellular domain(ECD) (FIGS. 5, 6). The hTβRII-hG1Fc fusion protein (R&D Systems) usedfor the screening described above incorporates the wild-typeTβRII_(short) isoform. In a follow-up analysis, the affinity of mGDF15binding to a fusion protein incorporating the wild-type TβRII_(long)isoform (R&D Systems) was found by surface plasmon resonance to be verysimilar to that of the fusion protein incorporating the TβRII_(short)isoform (K_(D)s at 37° C. were 2.7 nM and 4.8 nM, respectively). Havingobserved general equivalence of these short and long isoforms withregard to GDF15 binding, Applicants then generated a receptor-Fc fusionprotein consisting of the wild-type ECD of hTβRII_(short) (SEQ ID NO: 7)fused at its C-terminus with a human IgG2 Fc domain via a minimallinker. Unless noted otherwise, amino acid position numbering withregard to variants based on the TβRII short and long isoforms refers tothe corresponding position in the native precursors, SEQ ID NO: 5 andSEQ ID NO: 6, respectively.

Given the high-affinity binding of TβRII to GDF15, we tested whetherTβRII could be used as an inhibitor of GDF15. The fusion proteinhTβRII_(short)(23-159)-hG2Fc was tested in A549 cells transfected with areporter gene containing a CAGA-12 promoter construct and was found toinhibit hGDF15-induced gene activation in such cells with an IC₅₀ of0.15-0.5 nM. Potent inhibition of GDF15 signaling by the hTβRII_(short)ECD provides additional evidence that TβRII is the high-affinityreceptor for GDF15. Even though GDF15 exhibited no detectable binding toALK5 under cell-free conditions, suppression of endogenous ALK5 mRNA bysiRNA methodology markedly reduced mGDF15-mediated signaling in A549cells (a human pulmonary epithelial cell line) compared to controltreatment. In contrast, suppression of other type I receptors (ALK2,ALK3, ALK4, and ALK7) by siRNA methodology failed to alterGDF15-mediated signaling in A549 cells. This result indicates that theGDF15 ternary signaling complex includes ALK5 (TGFβ receptor type I) asits type I receptor and thus provides corroborating evidence for TβRIIas a functional type II receptor for GDF15.

Example 3. Generation of Receptor Fusion Protein Variants

TβRII ECD variants

Since TβRII also binds with high affinity to TGFβ1 and TGFβ3, nativeTβRII-Fc fusion protein affects signaling of these ligands as well asGDF15. While in some therapeutic settings this broader spectrum ofligand binding may be advantageous, in other settings a more selectivemolecule may be superior. Therefore, Applicants sought polypeptides withenhanced or reduced selectivity for GDF15 by generating fusion proteinscomprising variants of human TβRII ECD. The wild-typehTβRII_(short)(23-159) sequence shown below (SEQ ID NO: 7) served as thebasis for five receptor ECD variants listed below (SEQ ID NO: 8-12). Awild type hTβRII_(short)(23-159) was fused to an Fc portion of IgG2 togenerate a novel, base Fc fusion construct. See SEQ ID Nos. 50, 51 and52, below.

(SEQ ID NO: 7) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD  NQKSCMSNCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI  LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPD(1) The hTβRII_(short)(23-159/D110K) amino acid sequence shown below(SEQ ID NO: 8), in which the substituted residue is underlined.

(SEQ ID NO: 8) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD  NQKSCMSNCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHKFI  LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPD(2) The N-terminally truncated hTβRII_(short)(29-159) amino acidsequence shown below (SEQ ID NO: 9).

(SEQ ID NO: 9) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM  SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS  PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEYNTSNP D(3) The N-terminally truncated hTβRII_(short)(35-159) amino acidsequence shown below (SEQ ID NO: 10).

(SEQ ID NO: 10) 1  DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC 51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK  EKKKPGETFF101 MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRII_(short)(23-153) amino acidsequence shown below (SEQ ID NO: 11).

(SEQ ID NO: 11) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS 51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI  LEDAASPKCI101 MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y(5) The C-terminally truncated hTβRII_(short)(23-153/N70D) amino acidsequence shown below (SEQ ID NO: 12), in which the substituted residueis underlined.

(SEQ ID NO: 12) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSDCS 51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI  LEDAASPKCI101 MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y

Applicants also envision five corresponding variants (SEQ ID NO: 14-17)based on the wild-type hTβRII_(long)(23-184) sequence shown below (SEQID NO: 13), in which the 25 amino-acid insertion is underlined. Notethat splicing results in a conservative amino acid substitution(Val→Ile) at the flanking position C-terminal to the insertion. Sequencerelationships among several hTβRII_(short) variants and theirhTβRII_(long) counterparts are indicated in FIG. 7.

(SEQ ID NO: 13) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF 51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV  WRKNDENITL101 ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS  CSSDECNDNI 151IFSEEYNTSN PD(1) The hTβRII_(long)(23-184/D135K) amino acid sequence shown below (SEQID NO: 14), in which the substituted residue is double underlined.

(SEQ ID NO: 14) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF 51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV  WRKNDENITL101 ETVCHDPKLP YHKFILEDAA SPKCIMKEKK KPGETFFMCS  CSSDECNDNI 151IFSEEYNTSN PD(2) The N-terminally truncated hTβRII_(long)(29-184) amino acid sequenceshown below (SEQ ID NO: 15).

(SEQ ID NO: 15) 1 QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF51 CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY 151 NTSNPD(3) The N-terminally truncated hTβRII_(long)(60-184) amino acid sequenceshown below (same as SEQ ID NO: 10).

(same as SEQ ID NO: 10) 1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQKSCMSNCSIT SICEKPQEVC 51 VAVWRKNDEN ITLETVCHDP KLPYHDFILEDAASPKCIMK EKKKPGETFF 101 MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRII_(long)(23-178) amino acid sequenceshown below (SEQ ID NO: 16).

(SEQ ID NO: 16) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY(5) The C-terminally truncated hTβRII_(long)(23-178/N95D) amino acidsequence shown below (SEQ ID NO: 17), in which the substituted residueis double underlined.

(SEQ ID NO: 17) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSDCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY

Additional TβRII ECD variants include:

(A) The N- and C-terminally truncated hTβRII_(short)(35-153) orhTβRII_(long)(60-178) amino acid sequence shown below (SEQ ID NO: 47).

(SEQ ID NO: 47) 1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEY(B) The N- and C-terminally truncated hTβRII_(short)(29-153) amino acidsequence shown below (SEQ ID NO: 48).

(SEQ ID NO: 48) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEY(C) The N- and C-terminally truncated hTβRII_(long)(29-178) amino acidsequence shown below (SEQ ID NO: 49).

(SEQ ID NO: 49) 1 QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF51 CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEYAny of the above variants (SEQ ID NO: 8-12, 14-17, and 47-49) couldincorporate an insertion of 36 amino acids (SEQ ID NO: 18) between thepair of glutamate residues (positions 151 and 152 of SEQ ID NO: 5, orpositions 176 and 177 of SEQ ID NO: 6) located near the C-terminus ofthe hTβRII ECD, as occurs naturally in the hTβRII isoform C (Konrad etal., BMC Genomics 8:318, 2007).

(SEQ ID NO: 18) GRCKIRHIGS NNRLQRSTCQ NTGWESAHVM KTPGFRAs an example, the paired glutamate residues flanking the optionalinsertion site are denoted below (underlined) for thehTβRII_(short)(29-159) variant (SEQ ID NO: 9).

(SEQ ID NO: 9) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEYNTSNP D

Fc Domain Variants

hTβRII-hFc fusion proteins were generated in which five hTβRII_(short)variants described above were each fused at their C-terminus (via aminimal linker) to a human IgG2 Fc domain, which has the following aminoacid sequence (SEQ ID NO: 19):

(SEQ ID NO: 19) 1 VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK

Applicants envision hTβRII-hFc fusion proteins comprising alternative Fcdomains, including full-length human IgG1 Fc (hG1Fc) (SEQ ID NO: 20,below) and N-terminally truncated human IgG1 Fc (hG1Fc_(short)) (SEQ IDNO: 21, below). Optionally, a polypeptide unrelated to an Fc domaincould be attached in place of the Fc domain.

(SEQ ID NO: 20) 1 GGPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV51 DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 101NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS 151LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 201SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO: 21) 1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

Leader Sequence Variants

The following three leader sequences were considered:

(1) Native: (SEQ ID NO: 22) MGRGLLRGLWPLHIVLWTRIAS(2) Tissue plasminogen activator (TPA): (SEQ ID NO: 23)MDAMKRGLCCVLLLCGAVFVSP (3) Honey bee melittin (HBML): (SEQ ID NO: 24)MKFLVNVALVFMVVYISYIYAExpression of hTβRII-hFc Fusion Proteins

The selected hTβRII-hFc protein variants incorporate the TPA leader andhave the unprocessed amino-acid sequences shown in SEQ ID NOs: 25, 29,33, 37, and 41 (see Example 5). Corresponding nucleotide sequences forthese variants are SEQ ID NOs: 26, 30, 34, 38, and 42. SelectedhTβRII-hFc variants, each with a G2Fc domain (SEQ ID NO: 19), wereexpressed in HEK-293 cells and purified from conditioned media byfiltration and protein A chromatography. Purity of samples for reportergene assays was evaluated by SDS-PAGE and Western blot analysis.

Applicants envision additional hTβRII-hFc protein variants with theunprocessed amino-acid sequences shown in SEQ ID NOs: 27, 31, 35, 39,and 43, and corresponding nucleotide sequences shown in SEQ ID NOs: 28,32, 36, 40, and 44.

The amino acid sequence of the wild-type short constructhTβRII_(short)(23-159)-hG2Fc (SEQ ID NO: 50 is shown below.

(SEQ ID NO: 50) TIPPHVQKSV NNDMIVTDNN GAVKFPQLCKFCDVRFSTCD NQKSCMSNCS ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFILEDAASPKCI MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPDTGG GVECPPCPAPPVAGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE VHNAKTKPREEQFNSTFRVV SVLTVVHQDW LNGKEYKCKV SNKGLPAPIE KTISKTKGQP REPQVYTLPPSREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPMLDSDGS FFLYSKLTVDKSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

This protein was expressed from a construct including a TPA leadersequence, as shown below (SEQ ID NO:52). Dotted underline denotesleader, and solid underline denotes linker.

(SEQ ID NO: 52) 1

51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151FSEEYNTSNP DTGGGVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV 201TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV 251HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT 301KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK 351LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

The nucleic acid sequence encoding SEQ ID NO:52 is shown below:

(SEQ ID NO: 51) 1

51

101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA 351TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451TTCTCAGAAG AATATAACAC CAGCAATCCT GACACCGGTG GTGGAGTCGA 501GTGCCCACCG TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT 551TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC 601ACGTGCGTGG TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA 651CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG 701AGGAGCAGTT CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTCGTG 751CACCAGGACT GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA 801AGGCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC 851CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC 901AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ACCCCAGCGA 951CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA 1001CCACACCTCC CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG 1051CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC 1101CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC 1151TGTCTCCGGG TAAA

Example 4. Differential Ligand Inhibition by Receptor Fusion ProteinVariants in Cell-Based Assay

A reporter gene assay in A549 cells was used to determine the ability ofhTβRII-hFc variants to inhibit activity of GDF15, TGFβ1, TGFβ2, andTGFβ3. This assay is based on a human lung carcinoma cell linetransfected with a pGL3(CAGA)12 reporter plasmid (Dennler et al, 1998,EMBO 17: 3091-3100) as well as a Renilla reporter plasmid (pRLCMV) tocontrol for transfection efficiency. The CAGA motif is present in thepromoters of TGFβ-responsive genes (for example, PAI-1), so this vectoris of general use for factors signaling through SMAD2 and SMAD3.

On the first day of the assay, A549 cells (ATCC©: CCL-185™) weredistributed in 48-well plates at 6.5×10⁴ cells per well. On the secondday, a solution containing 10 μg pGL3(CAGA)12, 100 ng pRLCMV, 30 μlX-tremeGENE 9 (Roche Applied Science), and 970 μl OptiMEM (Invitrogen)was preincubated for 30 min, then added to Eagle's minimum essentialmedium (EMEM, ATCC©) supplemented with 0.1% BSA, which was applied tothe plated cells (500 μl/well) for incubation overnight at roomtemperature. On the third day, medium was removed, and cells wereincubated overnight at 37° C. with a mixture of ligands and inhibitorsprepared as described below.

Serial dilutions of test articles were made in a 48-well plate in a 200μl volume of assay buffer (EMEM+0.1% BSA). An equal volume of assaybuffer containing the test ligand was added to obtain a final ligandconcentration equal to the EC50 determined previously. Human GDF15 andmurine GDF15 were generated in-house (see above), while human TGFβ 1,human TGFβ2, and human TGFβ3 were obtained from PeproTech. Testsolutions were incubated at 37° C. for 30 minutes, then 250 μl of themixture was added to all wells. Each concentration of test article wasdetermined in duplicate. After incubation with test solutions overnight,cells were rinsed with phosphate-buffered saline, then lysed withpassive lysis buffer (Promega E1941) and stored overnight at −70° C. Onthe fourth and final day, plates were warmed to room temperature withgentle shaking. Cell lysates were transferred in duplicate to achemiluminescence plate (96-well) and analyzed in a luminometer withreagents from a Dual-Luciferase Reporter Assay system (Promega E1980) todetermine normalized luciferase activity.

This assay was used to screen receptor fusion protein variants forpotential inhibitory effects on cell signaling by TβRII ligands.Consistent with previous reports concerning wild-type TβRII_(short)-Fcand TβRII_(long)-Fc (del Re et al., J Biol Chem 279:22765, 2004), noneof the variants tested were able to inhibit TGFβ2, even at highconcentrations. However, hTβRII-hFc variants unexpectedly showeddifferential inhibition of cellular signaling mediated by GDF15, TGFβ1,and TGFβ3. Compared with wild-type TβRII_(short)(23-159)-G2Fc, theTβRII_(short)(23-159/D110K)-G2Fc variant exhibited potent inhibition ofGDF15 but loss of inhibition of TGFβ1 and greatly reduced inhibition(˜50 fold) of TGFβ3 (see table below). Position 110 is located in the“hook” region of TβRII (Radaev et al., J Biol Chem 285:14806, 2010) buthas not been suggested to confer selectivity among the recognized TβRIIligands TGFβ1, TGFβ2, and TGFβ3. Thus, this variant displays a profileof differential ligand inhibition in which GDF15 is inhibited mostpotently, TGFβ1 least potently, and TGFβ3 to an intermediate degree.

IC₅₀ (nM) mGDF15 hTGFβ1 hTGβ3 (35 (640 (270 Construct ng/ml) pg/ml)pg/ml) Full-length TβRII_(short)(23-159)- ~0.12 1.73 0.14 wild-type ECDG2Fc Full-length ECD with TβRII_(short)(23-159/ ~0.7 ND ~6.9 D110Ksubstitution D110K)-G2Fc (>73.6) ND, not determined

In a second experiment, potencies of variants with N-terminallytruncated TβRII ECD were compared with that of full-length wild-typeTβRII ECD. As shown in the table below, TβRII_(short)(29-159)-G2Fc andTβRII_(short)(35-159)-G2Fc displayed a greatly diminished ability toinhibit TGFβ3 but an undiminished (N′Δ6) or only slightly diminished(N′Δ12) ability to inhibit GDF15 compared to TβRII_(short)(23-159)-G2Fc(wild-type). Effects of N-terminal truncation on inhibition of TGFβ1compared to wild-type were intermediate in magnitude. Thus, these twovariants exhibit a profile of differential ligand inhibition in whichGDF15 is inhibited most potently, TGFβ3 least potently, and TGFβ1 to anintermediate degree.

IC₅₀ (nM) hGDF15 hTGFβ1 hTGFβ3 (70 or 112 (640 (270 Construct ng/ml)pg/ml) pg/ml) Full-length TβRII_(short)(23-159)-G2Fc 0.14-0.53 0.52 0.37wild-type ECD N'Δ6 ECD TβRII_(short)(29-159)-G2Fc 0.40 2.05 ND (>7.5)N'Δ12 ECD TβRII_(short)(35-159)-G2Fc 0.92 2.51 ND (>7.5) ND, notdetermined

In a third experiment, we determined the effect on potency of a N70Dsubstitution in a C-terminally truncated TβRII ECD. This aspartateresidue represents a potential glycosylation site. As shown in the tablebelow, TβRII_(short)(23-153/N70D)-G2Fc displayed greatly diminishedability to inhibit TGFβ1 and virtually undiminished ability to inhibitTGFβ3 compared to TβRII_(short)(23-153)-G2Fc. Effects of N70Dsubstitution on inhibition of GDF15 compared to bothTβRII_(short)(23-153)-G2Fc and wild-type were intermediate in magnitude.Thus, the C-terminally truncated variant with N70D substitution exhibitsa profile of differential ligand inhibition in which TGFβ3 is inhibitedmost potently, TGFβ1 least potently, and GDF15 to an intermediatedegree.

IC₅₀ (nM) hGDF15 hTGFβ1 hTGFβ3 (70 (640 (270 Construct ng/ml) pg/ml)pg/ml) Full-length TβRII_(short)(23-159)-G2Fc 0.14 ND ND wild-type ECDC'Δ6 ECD TβRII_(short)(23-153)-G2Fc 0.18 2.62 0.14 C'Δ6 ECD withTβRII_(short)(23-153/ 2.42 17.7 0.28 N70D substitution N70D)-G2Fc

Together, these results demonstrate that Applicants have generatedtruncations and mutations of the TβRII ECD that exhibit widely differentligand binding profiles. Notably, this demonstration reveals thatproperly expressed and purified GDF15 interacts directly with TβRII andcan be differentially inhibited by fusion proteins comprising variantsof the TβRII ECD. Activity profiles of these variants can be summarizedin the following table.

Summary of Ligand Selectivity Degree of Ligand Inhibition ConstructPotent Moderate Negligible Full-length wild-type TβRII_(short)(23-159)-GDF15 — TGFβ2 ECD G2Fc TGFβ1 TGFβ3 Full-length ECD withTβRII_(short)(23-159/ GDF15 TGFβ3 TGFβ1 D110K substitution D110K)-G2FcTGFβ2 N'Δ6 ECD TβRII_(short)(29-159)- GDF15 TGFβ1 TGFβ2 G2Fc TGFβ3 N'Δ12ECD TβRII_(short)(35-159)- GDF15 TGFβ1 TGFβ2 G2Fc TGFβ3 C'Δ6 ECD withTβRII_(short)(23-153/ TGFβ3 GDF15 TGFβ1 N70D substitution N70D)-G2FcTGFβ2

We predict that the TβRII_(long) ECD counterparts of these TβRII_(short)ECD variants will exhibit similar ligand selectivity. In addition, aC′Δ6 truncated ECD (such as SEQ ID NOs: 11 and 16 for the TβRII_(short)and TβRII_(long) isoforms, respectively) can be used as a base sequencefor TβRII_(short) or TβRII_(long) in which to introduce mutations andN-terminal truncations.

Example 5. Exemplary hTβRII-hFc Nucleic Acids and Proteins

This example summarizes nucleic acid constructs that can be used toexpress TβRII constructs in HEK-293 or CHO cells, according to themethods provided herein in order to provide the proteins isolated fromcell culture. In each case the mature protein isolated from cell culturewill have the leader sequence (dotted underline in each sequence below)removed.

Item 1 shows the amino acid sequence ofhTβRII_(short)(23-159/D110K)-hG2Fc (SEQ ID NO: 25). Double underlineindicates D110K substitution. Dotted underline denotes leader, and solidunderline denotes linker.

SEQ ID NO: 25) 1

51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVDHDPKLPY HKFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151FSEEYNTSNP DTGGGVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV 201TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV 251HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT 301KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK 351LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKItem 2 shows a nucleotide sequence encodinghTβRII_(short)(23-159/D110K)-hG2Fc (SEQ ID NO: 26). Double underlineindicates D110K substitution. Dotted underline denotes leader, and solidunderline denotes linker.

(SEQ ID NO: 26) 1

51

101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATAAGTTTA TTCTGGAAGA 351TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451TTCTCAGAAG AATATAACAC CAGCAATCCT GACACCGGTG GTGGAGTCGA 501GTGCCCACCG TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT 551TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC 601ACGTGCGTGG TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA 651CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG 701AGGAGCAGTT CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTCGTG 751CACCAGGACT GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA 801AGGCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC 851CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC 901AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ACCCCAGCGA 951CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA 1001CCACACCTCC CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG 1051CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC 1101CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC 1151TGTCTCCGGG TAAAItem 3 shows the amino acid sequence ofhTβRII_(short)(23-159/D110K)-hG1Fc_(short) (SEQ ID NO: 27). Doubleunderline indicates D110K substitution. Dotted underline denotes leader,and solid underline denotes linker.

(SEQ ID NO: 27) 1

51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVCHDPKLPY HKFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151FSEEYNTSNP DTGGGTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP   201EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT 251VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE 301MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY 351SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGKItem 4 shows a nucleotide sequence encodinghTβRII_(short)(23-159/D110K)-hG1Fc_(short) (SEQ ID NO: 28). Doubleunderline indicates D110K substitution. Dotted underline denotes leader,and solid underline denotes linker.

(SEQ ID NO: 28) 1

51

101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATAAGTTTA TTCTGGAAGA 351TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451TTCTCAGAAG AATATAACAC CAGCAATCCT GACACCGGTG GTGGAACTCA 501CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA CCGTCAGTCT 551TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT 601GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA 651GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC 701CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC 751GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC 801CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG 851GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG 901ATGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC 951CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT 1001ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAT 1051AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC 1101ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC 1151TCTCCCTGTC CCCGGGTAAAItem 5 shows the amino acid sequence of hTβRII_(short)(29-159)-hG2Fc(SEQ ID NO: 29). Dotted underline denotes leader, and solid underlinedenotes linker.

(SEQ ID NO: 29) 1

51 DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC VAVWRKNDEN ITLETVCHDP 101KLPYHDFILE DAASPKCIMK EKKKPGETFF MCSCSSDECN DNIIFSEEYN 151TSNPDTGGGV ECPPCPAPPV AGPSVFLFPP KPKDTLMISR TPEVTCVVVD 201VSHEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTFRVVSV LTVVHQDWLN 251GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL   301TCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFF LYSKLTVDKS 351RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKItem 6 shows a nucleotide sequence encoding hTβRII_(short)(29-159)-hG2Fc(SEQ ID NO: 30). Dotted underline denotes leader, and solid underlinedenotes linker.

(SEQ ID NO: 30)

 101 TCACTGACAA CAACGGTGCA GTCAAGTTTC CACAACTGTG TAAATTTTGT 151 GATGTGAGAT TTTCCACCTG TGACAACCAG AAATCCTGCA TGAGCAACTG 201 CAGCATCACC TCCATCTGTG AGAAGCCACA GGAAGTCTGT GTGGCTGTAT 251 GGAGAAAGAA TGACGAGAAC ATAACACTAG AGACAGTTTG CCATGACCCC 301 AAGCTCCCCT ACCATGACTT TATTCTGGAA GATGCTGCTT CTCCAAAGTG 351 CATTATGAAG GAAAAAAAAA AGCCTGGTGA GACTTTCTTC ATGTGTTCCT 401 GTAGCTCTGA TGAGTGCAAT GACAACATCA TCTTCTCAGA AGAATATAAC 451 ACCAGCAATC CTGACACCGG TGGTGGAGTC GAGTGCCCAC CGTGCCCAGC 501 ACCACCTGTG GCAGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 551 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACGTGCGT GGTGGTGGAC 601 GTGAGCCACG AAGACCCCGA GGTCCAGTTC AACTGGTACG TGGACGGCGT 651 GGAGGTGCAT AATGCCAAGA CAAAGCCACG GGAGGAGCAG TTCAACAGCA 701 CGTTCCGTGT GGTCAGCGTC CTCACCGTCG TGCACCAGGA CTGGCTGAAC 751 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGGCCTCC CAGCCCCCAT 801 CGAGAAAACC ATCTCCAAAA CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 851 ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 901 ACCTGCCTGG TCAAAGGCTT CTACCCCAGC GACATCGCCG TGGAGTGGGA 951 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACACCT CCCATGCTGG1001 ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT GGACAAGAGC1051 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT1101 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAAItem 7 shows the amino acid sequence ofhTβRII_(short)(29-159)-hG1Fc_(short) (SEQ ID NO: 31). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 31)

 51 DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC VAVWRKNDEN ITLETVCHDP101 KLPYHDFILE DAASPKCIMK EKKKPGETFF MCSCSSDECN DNIIFSEEYN151 TSNPDTGGGT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV201 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW251 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV301 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD351 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKItem 8 shows a nucleotide sequence encodinghTβRII_(short)(29-159)-hG1Fc_(short) (SEQ ID NO: 32). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 32)

 101 TCACTGACAA CAACGGTGCA GTCAAGTTTC CACAACTGTG TAAATTTTGT 151 GATGTGAGAT TTTCCACCTG TGACAACCAG AAATCCTGCA TGAGCAACTG 201 CAGCATCACC TCCATCTGTG AGAAGCCACA GGAAGTCTGT GTGGCTGTAT 251 GGAGAAAGAA TGACGAGAAC ATAACACTAG AGACAGTTTG CCATGACCCC 301 AAGCTCCCCT ACCATGACTT TATTCTGGAA GATGCTGCTT CTCCAAAGTG 351 CATTATGAAG GAAAAAAAAA AGCCTGGTGA GACTTTCTTC ATGTGTTCCT 401 GTAGCTCTGA TGAGTGCAAT GACAACATCA TCTTCTCAGA AGAATATAAC 451 ACCAGCAATC CTGACACCGG TGGTGGAACT CACACATGCC CACCGTGCCC 501 AGCACCTGAA CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC 551 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 601 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA 651 CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 701 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 751 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 801 CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC 851 AGGTGTACAC CCTGCCCCCA TCCCGGGAGG AGATGACCAA GAACCAGGTC 901 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 951 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG1001 TGCTGGACTC CGACGGCTCC TTCTTCCTCT ATAGCAAGCT CACCGTGGAC1051 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA1101 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCCCCGGGTA 1151 AAItem 9 shows the amino acid sequence of hTβRII_(short)(35-159)-hG2Fc(SEQ ID NO: 33). Dotted underline denotes leader, and solid underlinedenotes linker.

(SEQ ID NO: 33)

 51 CDNQKSCMSN CSITSICEKP QEVCVAVWRK NDENITLETV CHDPKLPYHD101 FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFS EEYNTSNPDT151 GGGVECPPCP APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP201 EVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC251 KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG301 FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN351 VFSCSVMHEA LHNHYTQKSL SLSPGKItem 10 shows a nucleotide sequence encodinghTβRII_(short)(35-159)-hG2Fc (SEQ ID NO: 34). Dotted underline denotesleader, and solid underline denotes linker.

(SEQ ID NO: 34)

 101 CAGTCAAGTT TCCACAACTG TGTAAATTTT GTGATGTGAG ATTTTCCACC 151 TGTGACAACC AGAAATCCTG CATGAGCAAC TGCAGCATCA CCTCCATCTG 201 TGAGAAGCCA CAGGAAGTCT GTGTGGCTGT ATGGAGAAAG AATGACGAGA 251 ACATAACACT AGAGACAGTT TGCCATGACC CCAAGCTCCC CTACCATGAC 301 TTTATTCTGG AAGATGCTGC TTCTCCAAAG TGCATTATGA AGGAAAAAAA 351 AAAGCCTGGT GAGACTTTCT TCATGTGTTC CTGTAGCTCT GATGAGTGCA 401 ATGACAACAT CATCTTCTCA GAAGAATATA ACACCAGCAA TCCTGACACC 451 GGTGGTGGAG TCGAGTGCCC ACCGTGCCCA GCACCACCTG TGGCAGGACC 501 GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC 551 GGACCCCTGA GGTCACGTGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCC 601 GAGGTCCAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA 651 GACAAAGCCA CGGGAGGAGC AGTTCAACAG CACGTTCCGT GTGGTCAGCG 701 TCCTCACCGT CGTGCACCAG GACTGGCTGA ACGGCAAGGA GTACAAGTGC 751 AAGGTCTCCA ACAAAGGCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA 801 AACCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC 851 GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC 901 TTCTACCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA 951 GAACAACTAC AAGACCACAC CTCCCATGCT GGACTCCGAC GGCTCCTTCT1001 TCCTCTACAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC1051 GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA1101 GAAGAGCCTC TCCCTGTCTC CGGGTAAAItem 11 shows the amino acid sequence ofhTβRII_(short)(35-159)-hG1Fc_(short) (SEQ ID NO: 35). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 35)

 51 CDNQKSCMSN CSITSICEKP QEVCVAVWRK NDENITLETV CHDPKLPYHD101 FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFS EEYNTSNPDT151 GGGTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE201 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY251 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV301 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ351 GNVFSCSVMH EALHNHYTQK SLSLSPGKItem 12 shows a nucleotide sequence encodinghTβRII_(short)(35-159)-hG1Fc_(short) (SEQ ID NO: 36). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 36)

 101 CAGTCAAGTT TCCACAACTG TGTAAATTTT GTGATGTGAG ATTTTCCACC 151 TGTGACAACC AGAAATCCTG CATGAGCAAC TGCAGCATCA CCTCCATCTG 201 TGAGAAGCCA CAGGAAGTCT GTGTGGCTGT ATGGAGAAAG AATGACGAGA 251 ACATAACACT AGAGACAGTT TGCCATGACC CCAAGCTCCC CTACCATGAC 301 TTTATTCTGG AAGATGCTGC TTCTCCAAAG TGCATTATGA AGGAAAAAAA 351 AAAGCCTGGT GAGACTTTCT TCATGTGTTC CTGTAGCTCT GATGAGTGCA 401 ATGACAACAT CATCTTCTCA GAAGAATATA ACACCAGCAA TCCTGACACC 451 GGTGGTGGAA CTCACACATG CCCACCGTGC CCAGCACCTG AACTCCTGGG 501 GGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 551 TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 601 GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA 651 TGCCAAGACA AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG 701 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC 751 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT 801 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC 851 CATCCCGGGA GGAGATGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC 901 AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 951 GCCGGAGAAC AACTACAAGA CCACGCCTCC CGTGCTGGAC TCCGACGGCT1001 CCTTCTTCCT CTATAGCAAG CTCACCGTGG ACAAGAGCAG GTGGCAGCAG1051 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA1101 CACGCAGAAG AGCCTCTCCC TGTCCCCGGG TAAAItem 13 shows the amino acid sequence of hTβRII_(short)(23-153)-hG2Fc(SEQ ID NO: 37). Dotted underline denotes leader, and solid underlinedenotes linker.

(SEQ ID NO: 37)

 51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII151 FSEEYTGGGV ECPPCPAPPV AGPSVFLFPP KPKDTLMISR TPEVTCVVVD201 VSHEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTFRVVSV LTVVHQDWLN251 GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL301 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFF LYSKLTVDKS351 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKItem 14 shows a nucleotide sequence encodinghTβRII_(short)(23-153)-hG2Fc (SEQ ID NO: 38). Dotted underline denotesleader, and solid underline denotes linker.

(SEQ ID NO: 38)

 101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151 CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201 ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251 AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA 351 TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401 CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451 TTCTCAGAAG AATATACCGG TGGTGGAGTC GAGTGCCCAC CGTGCCCAGC 501 ACCACCTGTG GCAGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 551 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACGTGCGT GGTGGTGGAC 601 GTGAGCCACG AAGACCCCGA GGTCCAGTTC AACTGGTACG TGGACGGCGT 651 GGAGGTGCAT AATGCCAAGA CAAAGCCACG GGAGGAGCAG TTCAACAGCA 701 CGTTCCGTGT GGTCAGCGTC CTCACCGTCG TGCACCAGGA CTGGCTGAAC 751 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGGCCTCC CAGCCCCCAT 801 CGAGAAAACC ATCTCCAAAA CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 851 ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 901 ACCTGCCTGG TCAAAGGCTT CTACCCCAGC GACATCGCCG TGGAGTGGGA 951 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACACCT CCCATGCTGG1001 ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT GGACAAGAGC1051 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT1101 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAAItem 15 shows the amino acid sequence ofhTβRII_(short)(23-153)-hG1Fc_(short) (SEQ ID NO: 39). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 39)

 51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII151 FSEEYTGGGT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV201 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW251 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV301 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD351 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKItem 16 shows a nucleotide sequence encodinghTβRII_(short)(23-153)-hG1Fc_(short) (SEQ ID NO: 40). Dotted underlinedenotes leader, and solid underline denotes linker.

(SEQ ID NO: 40)

 101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151 CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201 ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251 AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA 351 TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401 CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451 TTCTCAGAAG AATATACCGG TGGTGGAACT CACACATGCC CACCGTGCCC 501 AGCACCTGAA CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC 551 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 601 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA 651 CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 701 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 751 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 801 CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC 851 AGGTGTACAC CCTGCCCCCA TCCCGGGAGG AGATGACCAA GAACCAGGTC 901 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 951 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG1001 TGCTGGACTC CGACGGCTCC TTCTTCCTCT ATAGCAAGCT CACCGTGGAC1051 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA1101 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCCCCGGGTA 1151 AAItem 17 shows the amino acid sequence ofhTβRII_(short)(23-153/N70D)-hG2Fc (SEQ ID NO: 41). Double underlineindicates N70D substitution. Dotted underline denotes leader, and solidunderline denotes linker.

(SEQ ID NO: 41)

 51 QLCKFCDVRF STCDNQKSCM SDCSITSICE KPQEVCVAVW RKNDENITLE101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII151 FSEEYTGGGV ECPPCPAPPV AGPSVFLFPP KPKDTLMISR TPEVTCVVVD201 VSHEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTFRVVSV LTVVHQDWLN251 GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL301 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFF LYSKLTVDKS351 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKItem 18 shows a nucleotide sequence encodinghTβRII_(short)(23-153/N70D)-hG2Fc (SEQ ID NO: 42). Double underlineindicates N70D substitution. Dotted underline denotes leader, and solidunderline denotes linker.

(SEQ ID NO: 42)

 101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA  151 CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA  201 ATCCTGCATG AGCGACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG  251 AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG  301 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA  351 TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA  401 CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC  451 TTCTCAGAAG AATATACCGG TGGTGGAGTC GAGTGCCCAC CGTGCCCAGC  501 ACCACCTGTG GCAGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG  551 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACGTGCGT GGTGGTGGAC  601 GTGAGCCACG AAGACCCCGA GGTCCAGTTC AACTGGTACG TGGACGGCGT  651 GGAGGTGCAT AATGCCAAGA CAAAGCCACG GGAGGAGCAG TTCAACAGCA  701 CGTTCCGTGT GGTCAGCGTC CTCACCGTCG TGCACCAGGA CTGGCTGAAC  751 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGGCCTCC CAGCCCCCAT  801 CGAGAAAACC ATCTCCAAAA CCAAAGGGCA GCCCCGAGAA CCACAGGTGT  851 ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG  901 ACCTGCCTGG TCAAAGGCTT CTACCCCAGC GACATCGCCG TGGAGTGGGA  951 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACACCT CCCATGCTGG 1001 ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT GGACAAGAGC 1051 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT 1101 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA Item 19 shows the amino acid sequence ofhTβRII_(short)(23-153/N70D)-hG1Fc_(short) (SEQ ID NO: 43). Doubleunderline indicates N70D substitution. Dotted underline denotes leader,and solid underline denotes linker.

(SEQ ID NO: 43)

 51 QLCKFCDVRF STCDNQKSCM SDCSITSICE KPQEVCVAVW RKNDENITLE101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII151 FSEEYTGGGT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV201 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW251 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV301 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD351 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKItem 20 shows a nucleotide sequence encodinghTβRII_(short)(23-153/N70D)-hG1Fc_(short) (SEQ ID NO: 44). Doubleunderline indicates N70D substitution. Dotted underline denotes leader,and solid underline denotes linker.

(SEQ ID NO: 44)

 101 TTAATAACGA CATGATAGTC ACTGACAACA ACGGTGCAGT CAAGTTTCCA 151 CAACTGTGTA AATTTTGTGA TGTGAGATTT TCCACCTGTG ACAACCAGAA 201 ATCCTGCATG AGCGACTGCA GCATCACCTC CATCTGTGAG AAGCCACAGG 251 AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 301 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA 351 TGCTGCTTCT CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA 401 CTTTCTTCAT GTGTTCCTGT AGCTCTGATG AGTGCAATGA CAACATCATC 451 TTCTCAGAAG AATATACCGG TGGTGGAACT CACACATGCC CACCGTGCCC 501 AGCACCTGAA CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC 551 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 601 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA 651 CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 701 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 751 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 801 CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC 851 AGGTGTACAC CCTGCCCCCA TCCCGGGAGG AGATGACCAA GAACCAGGTC 901 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 951 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG1001 TGCTGGACTC CGACGGCTCC TTCTTCCTCT ATAGCAAGCT CACCGTGGAC1051 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA1101 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCCCCGGGTA 1151 AAItem 21 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-159/D110K)-hG2Fc (SEQ ID NO: 53). Doubleunderline indicates D110K substitution. Single underline denotes linker.

(SEQ ID NO: 53)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HKFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYNTSNP DTGGGVECPP CPAPPVAGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYVDGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEYKCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKItem 22 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-159/D110K)-hG1Fc_(short) (SEQ ID NO: 54).Double underline indicates D110K substitution. Single underline denoteslinker.

(SEQ ID NO: 54)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HKFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYNTSNP DTGGGTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Item 23 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(29-159)-hG2Fc (SEQ ID NO: 55). Singleunderline denotes linker.

(SEQ ID NO: 55)     QKSVNN DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQKSCMSNCSIT SICEKPQEVC VAVWRKNDEN ITLETVCHDPKLPYHDFILE DAASPKCIMK EKKKPGETFF MCSCSSDECNDNIIFSEEYN TSNPDTGGGV ECPPCPAPPV AGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVQF NWYVDGVEVHNAKTKPREEQ FNSTFRVVSV LTVVHQDWLN GKEYKCKVSNKGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKItem 24 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(29-159)-hG1Fc_(short) (SEQ ID NO: 56).Single underline denotes linker.

(SEQ ID NO: 56)     QKSVNN DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQKSCMSNCSIT SICEKPQEVC VAVWRKNDEN ITLETVCHDPKLPYHDFILE DAASPKCIMK EKKKPGETFF MCSCSSDECNDNIIFSEEYN TSNPDTGGGT HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKItem 25 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(35-159)-hG2Fc (SEQ ID NO: 57). Singleunderline denotes linker.

(SEQ ID NO: 57)     DMIVTD NNGAVKFPQL CKFCDVRFST CDNQKSCMSNCSITSICEKP QEVCVAVWRK NDENITLETV CHDPKLPYHDFILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFSEEYNTSNPDT GGGVECPPCP APPVAGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKPREEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPIEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKItem 26 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(35-159)-hG1Fc_(short) (SEQ ID NO: 58).Single underline denotes linker.

(SEQ ID NO: 58)     DMIVTD NNGAVKFPQL CKFCDVRFST CDNQKSCMSNCSITSICEKP QEVCVAVWRK NDENITLETV CHDPKLPYHDFILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFSEEYNTSNPDT GGGTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKTKPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSKLTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKItem 27 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-153)-hG2Fc (SEQ ID NO: 59). Singleunderline denotes linker.

(SEQ ID NO: 59)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYTGGGV ECPPCPAPPV AGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVQF NWYVDGVEVHNAKTKPREEQ FNSTFRVVSV LTVVHQDWLN GKEYKCKVSNKGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

Item 28 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-153)-hG1Fc_(short) (SEQ ID NO: 60).Single underline denotes linker.

(SEQ ID NO: 60)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYTGGGT HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Item 29 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-153/N70D)-hG2Fc (SEQ ID NO: 61). Doubleunderline indicates N70D substitution. Single underline denotes linker.

(SEQ ID NO: 61)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SDCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYTGGGV ECPPCPAPPV AGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVQF NWYVDGVEVHNAKTKPREEQ FNSTFRVVSV LTVVHQDWLN GKEYKCKVSNKGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKItem 30 shows the mature amino acid sequence (i.e., without the leadersequence) of hTβRII_(short)(23-153/N70D)-hG1Fc_(short) (SEQ ID NO: 62).Double underline indicates N70D substitution. Single underline denoteslinker.

(SEQ ID NO: 62)     TIPPHV QKSVNNDMIV TDNNGAVKFP QLCKFCDVRFSTCDNQKSCM SDCSITSICE KPQEVCVAVW RKNDENITLETVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSCSSDECNDNII FSEEYTGGGT HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1-16. (canceled)
 17. A method of identifying functional mutants of aTβRII polypeptide, comprising: a. producing a combinatorial library ofgenes encoding variant polypeptides; and b. evaluating the ability ofeach variant polypeptide to bind to Transforming Growth Factor β1 andTransforming Growth Factor β3.
 18. The method of claim 17, wherein thelibrary of genes is produced by enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences.
 19. The method of claim17 or claim 18, wherein the variant polypeptide sequences can beexpressed individually.
 20. The method of claim 17 or claim 18, whereinthe variant polypeptide sequences can be expressed as a set of largerfusion proteins.
 21. The method of claim 17 or claim 18, wherein thevariant polypeptides are truncation mutants.
 22. The method of claim 17or claim 18, wherein the variant polypeptides are combinatorial mutantsof a TβRII polypeptide.
 23. The method of claim 17 or claim 18, whereineach gene in the library of genes includes at least a portion of apotential TβRII polypeptide sequence.
 24. The method of claim 17 orclaim 18, wherein a nucleotide sequence encoding the gene is chemicallysynthesized.
 25. The method of claim 17, wherein the chemicallysynthesized gene sequence is ligated into a vector for expression. 26.The method of claim 17 or claim 18, wherein the combinatorial library isproduced using alanine scanning mutagenesis, linker scanningmutagenesis, saturation mutagenesis, PCR mutagenesis, or by randommutagenesis.